Psma-targeting compounds and uses thereof

ABSTRACT

Compositions and methods for visualizing tissue under illumination with near-infrared radiation, including compounds comprising near-infrared, closed chain, sulfo-cyanine dyes and prostate specific membrane antigen ligands are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/474,497, filed Sep. 14, 2021, which is a continuation of U.S. patentapplication Ser. No. 17/331,113, filed May 26, 2021, which is acontinuation of U.S. patent application Ser. No. 16/704,137, filed Dec.5, 2019, now abandoned, which is a continuation of U.S. patentapplication Ser. No. 15/618,788, filed Jun. 9, 2017, now abandoned,which claims the benefit of U.S. Provisional Application No. 62/324,097,filed Apr. 18, 2016, and is a continuation-in-part of U.S. patentapplication Ser. No. 14/243,535, filed Apr. 2, 2014, now U.S. Pat. No.9,776,977 issued Oct. 3, 2017, which is a divisional of U.S. patentapplication Ser. No. 13/257,499 filed Sep. 19, 2011, and now U.S. Pat.No. 9,056,841 issued Jun. 16, 2015, which is a 35 U.S.C. § 371 NationalStage Entry of International Application No. PCT/US2010/028020 having aninternational filing date of Mar. 19, 2010, which claims the benefit ofU.S. Provisional Application No. 61/248,934 filed Oct. 6, 2009, U.S.Provisional Application No. 61/248,067 filed Oct. 2, 2009, U.S.Provisional Application No. 61/161,484 filed Mar. 19, 2009, and U.S.Provisional Application No. 61/161,485 filed Mar. 19, 2009, each ofwhich is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENTAL INTEREST

This invention was made with government support under CA092871 awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND

Prostate cancer (PCa) is the most commonly diagnosed malignancy and thesecond leading cause of cancer-related death in men in the UnitedStates. Prostate-specific membrane antigen (PSMA) is a marker forandrogen-independent disease that also is expressed on solid(nonprostate) tumor neovasculature. Complete detection and eradicationof primary tumor and metastatic foci are required to effect a cure inpatients with cancer.

SUMMARY

In some aspects, the presently disclosed subject matter provides acompound having the structure:

wherein:

Z is tetrazole or CO₂Q;

each Q is independently selected from hydrogen or a protecting group;

a is 1, 2, 3, or 4;

R is each independently H or C₁-C₄ alkyl;

Ch is a metal chelating moiety optionally including a chelated metal,wherein Ch optionally includes any additional atoms or linkers necessaryto attach the metal chelating moiety to the rest of the compound;

W is —NRC(O)—, —NRC(O)NR—, NRC(S)NR—, —NRC(O)O—, —OC(O)NR—, —OC(O)—,—C(O)NR—, or —C(O)O—;

Y is —C(O)—, —NRC(O)—, —NRC(S)—, —OC(O);

V is —C(O)—, —NRC(O)—, —NRC(S)—, or —OC(O)—;

m is 1, 2, 3, 4, 5, or 6;

n is 1, 2, 3, 4, 5 or 6;

p is 0, 1, 2, or 3, and when p is 2 or 3, each R¹ may be the same ordifferent;

R¹ is H, C₁-C₆ alkyl, C₂-C₁₂ aryl, or C₄-C₁₆ alkylaryl;

R² and R³ are independently H, CO₂H, or CO₂R⁴, wherein R⁴ is a C₁-C₆alkyl, C₂-C₁₂ aryl, or C₄-C₁₆ alkylaryl, wherein when one of R² and R³is CO₂H or CO₂R⁴, the other is H, and when p is 0, one of R² and R³ isCO₂R⁴, and the other is H; and

pharmaceutically acceptable salts thereof.

In some aspects, the compound of formula (I) has the structure:

In some aspects of the compound of formula (I), Z is CO₂Q; each Q ishydrogen; R is H; a is 4; m is 6; n is 3; p is 2; R¹ is C₂-C₁₂ aryl,wherein the aryl may be substituted or unsubstituted and R¹ may be thesame or different; R² is CO₂H; R³ is H; W is —NRC(O)—, wherein R is H; Vis —C(O)—; and Ch includes any additional atoms or linkers necessary toattach the metal chelating moiety to the rest of the compound.

In certain aspects, R¹ is phenyl or a substituted phenyl. In particularaspects, R¹ is a phenyl substituted at 1, 2, 3, or 4 positions with asubstituent group selected from the group consisting of halogen, cyano,hydroxyl, nitro, azido, amino, alkanoyl, carboxamido, alkyl, alkenyl,alkynyl, alkoxy, aryloxy, alkylthio, alkylsulfinyl, alkylsulfonyl,aminoalkyl, carbocyclic aryl, arylalkyl, arylalkoxy, and a saturated,unsaturated, or aromatic heterocyclic group, which may be furthersubstituted. In more particular aspects, R¹ is a phenyl substituted witha halogen and a hydroxyl.

In some aspects, the additional atoms or linkers necessary to attach themetal chelating moiety to the rest of the compound comprises an alkyl,aryl, combination of alkyl and aryl, or alkyl and aryl groups havingheteroatoms. In certain aspects, the additional atoms or linkersnecessary to attach the metal chelating moiety to the rest of thecompound comprises an alkyl, wherein the alkyl may be substituted orunsubstituted.

In some aspects, Ch comprises a structure selected from the groupconsisting of:

In particular aspects, Ch comprises1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

In some aspects, the compound of formula (I) is selected from the groupconsisting of:

In some aspects, Ch includes a chelated metal and the chelated metalcomprises a radioactive isotope. In certain aspects, the radioactiveisotope is Tc-99m, In-111, Ga-67, Ga-68, Y-86, Y-90, Lu-177, Re-186,Re-188, Cu-64, Cu-67, Co-55, Co-57, Sc-47, Ac-225, Bi-213, Bi-212,Pb-212, Sm-153, Ho-166, or Dy-166. In particular aspects, theradioactive isotope is Ga-68 or Lu-177.

In other aspects, the presently disclosed subject matter provides amethod for imaging one or more prostate-specific membrane antigen (PSMA)tumors, or cells the method comprising contacting the one or moretumors, or cells, with an effective amount of a compound of formula (I),or a pharmaceutically acceptable salt thereof, and making an image.

In some aspects, the imaging comprises positron emission tomography(PET).

In some aspects, the one or more PSMA-expressing tumors or cells isselected from the group consisting of a prostate tumor or cell, ametastasized prostate tumor or cell, a lung tumor or cell, a renal tumoror cell, a glioblastoma, a pancreatic tumor or cell, a bladder tumor orcell, a sarcoma, a melanoma, a breast tumor or cell, a colon tumor orcell, a germ cell, a pheochromocytoma, an esophageal tumor or cell, astomach tumor or cell, and combinations thereof.

In other aspects, the presently disclosed subject matter provides amethod for treating a tumor comprising administering a therapeuticallyeffective amount of a compound of formula (I), or a pharmaceuticallyacceptable salt thereof, wherein the compound includes a therapeuticallyeffective radioisotope.

In other aspects, the presently disclosed subject matter provides a kitcomprising a compound of formula (I), or a pharmaceutically acceptablesalt thereof.

Certain aspects of the presently disclosed subject matter having beenstated hereinabove, which are addressed in whole or in part by thepresently disclosed subject matter, other aspects will become evident asthe description proceeds when taken in connection with the accompanyingExamples and Figures as best described herein below.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

Having thus described the presently disclosed subject matter in generalterms, reference will now be made to the accompanying Figures, which arenot necessarily drawn to scale, and wherein:

FIG. 1 shows whole body and ex vivo organ imaging of mouse with PSMA⁺PC3 PIP tumor and PSMA⁻ PC3 flu tumor at 24 h postinjection of 1 nmol ofDyLight800-3;

FIG. 2 shows the absorbance and emission spectra, and quantum yield ofexemplary compound YC-27;

FIG. 3 shows the fluorescence decay of exemplary compound YC-27;

FIG. 4 shows an IC₅₀ curve of compound YC-27 using a fluorescence-basedNAALADase assay;

FIG. 5A-FIG. 5O show in vivo imaging of a NOD/SCID mouse (mouse #1),bearing PC3-PIP (forward left flank) and PC3-flu (forward right flank)tumors. Mouse #1 received 10 nmol of YC-27 and dorsal (animal prone) andventral (animal supine) views were obtained. Dorsal and ventral views at40 min p.i. (FIG. 5A, FIG. 5B, respectively); 18.5 h (FIG. 5C, FIG. 5D);23 h (FIG. 5E, FIG. 5F); 42.5 h (FIG. 5G, FIG. 5H); 68 h (FIG. 5I, FIG.5J). Dorsal view of pre-injection image (FIG. 5K). Dorsal and ventralviews 70.5 h p.i. (FIG. 5L, FIG. 5M). Images after midline laparotomy(FIG. 5N) and individually harvested organs (FIG. 5O) on a Petri dish at70.5 h p.i. Images were scaled to the same maximum (arbitrary units);

FIG. 6A-FIG. 6T show in vivo imaging of a NOD/SCID mouse (mouse #2)(left panel), bearing PC3-PIP (forward left flank) and PC3-flu (forwardright flank) tumors. Mouse #2 received 1 nmol of YC-27 and dorsal(animal prone) and ventral (animal supine) views were obtained. Dorsaland ventral views of the pre-injection image (FIG. 6A, FIG. 6B,respectively); 10 min p.i. (FIG. 6C, FIG. 6D); 20.5 h (FIG. 6E, FIG.6F); 24 h (FIG. 6G, FIG. 6H). Images after midline laparotomy (FIG. 6I)and individually harvested organs (FIG. 6J) on a Petri dish at 24 h p.i.Right Panels: Mouse #3 in same orientation as mouse #2. Mouse #3received 1 nmol of YC-27 co-injected with 1 μmol of DCIBzL, which servedas a blocking agent to test binding specificity. Images were scaled tothe same maximum (arbitrary units);

FIG. 7 shows PC3-PIP and PC3-flu cells treated with fluorescent compoundYC-VIII-36 (green, top left) and DAPI (blue), and PC3-PIP and PC3-flucells treated with both YC-VIII-36 and PSMA inhibitor, PMPA;

FIG. 8 shows PC3-PIP cells treated with DAPI (blue) and varyingconcentrations of YC-VIII-36 (green);

FIG. 9 shows time dependent internalization of YC-VIII-36 into PC3-PIPcells treated with YC-VIII-36 (green) and DAPI (blue);

FIG. 10 shows titration and detection of varying amounts of YC-VIII-36injected subcutaneously into a nude mouse. (IVIS spectrum with 10 secondexposure followed by spectral unmixing);

FIG. 11 shows fluorescence images of a PSMA+ PC3-PIP and PSMA− PC3-flutumor-bearing mouse injected intravenously with exemplary compoundYC-VIII-36;

FIG. 12 shows fluorescence images of a PSMA+ PC3-PIP and PSMA− PC3-flutumor-bearing mouse injected intravenously with exemplary compoundYC-VIII-36 180 minutes after injection (top) and biodistribution ofexemplary compound YC-VIII-36 180 minutes after injection (bottom);

FIG. 13 shows FACS analysis showing the percent subpopulation of PSMApositive cells in PC3-flu, PC3-PIP, and LNCaP cells;

FIG. 14 shows cell sorting results for PC3-PIP cells treated withexemplary compound YC-VIII-36, including initial percentage (topcenter), and after 3 passages of sorting (bottom);

FIG. 15A-FIG. 15C show the number of spiked PIP-pos cells into 10million of PC3-flu detectable by 100 nM compound YC-VIII-36 in flowcytometry (BD LSR-II). Gate P1 is total number of single cells counted;gate P2 at higher intensity is the number of Pip-pos cells detected andgate P3 at lower intensity;

FIG. 16 shows SPECT-CT images of a PSMA+LNCaP tumor-bearing mouseinjected intravenously with exemplary compound [^(99m)Tc]SRV32:

FIG. 17. GE eXplore VISTA pseudodynamic PET image (co-registered withthe corresponding CT image) of a PSMA+LNCaP tumor-bearing mouse injectedintravenously with 0.2 mCi (7.4 MBq) of exemplary compound [⁶⁸Ga]SRV27;

FIG. 18. GE eXplore VISTA PET image (co-registered with thecorresponding CT image) of a PSMA+PIP and PSMA− flu tumor-bearing mouseinjected intravenously with 0.2 mCi (7.4 MBq) of exemplary compound[⁶⁸Ga]SRV100:

FIG. 19 shows a synthetic scheme for exemplary compound SRV100 and[¹¹¹In]SRV100:

FIG. 20 shows SPECT-CT images of a PSMA+ PC-3 PIP tumor-bearing mouseinjected intravenously with exemplary compound [¹¹¹In]SRV27;

FIG. 21 shows SPECT-CT images of a PSMA+ PC-3 PIP tumor-bearing mouseinjected intravenously with exemplary compound [¹¹¹In]SRV100;

FIG. 22 shows SPECT-CT images of a PSMA+ PC-3 PIP tumor-bearing mouseinjected intravenously with exemplary dual modality compound[¹¹¹In]SRV73;

FIG. 23 shows SPECT-CT images of a PSMA+LNCaP tumor-bearing mouseinjected intravenously with exemplary compound [¹¹¹Tc]SRVI34B;

FIG. 24 shows SPECT-CT images of a PSMA+ PC3-PIP tumor-bearing mouseinjected intravenously with exemplary compound [¹¹¹Tc]SRVI34B;

FIG. 25 shows SPECT-CT images of a PSMA+ PC3-PIP (forward left flank)and PSMA− PC3-flu (forward right flank) tumor-bearing mouse injectedintravenously with exemplary compound [^(99m)Tc]SRV134A; and

FIG. 26 shows SPECT-CT images of a PSMA+ PC3-PIP (forward left flank)and PSMA− PC3-flu (forward right flank) tumor-bearing mouse injectedintravenously with exemplary compound [^(99m)Tc]SRVI34B.

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fullyhereinafter with reference to the accompanying Figures, in which some,but not all embodiments of the inventions are shown. Like numbers referto like elements throughout. The presently disclosed subject matter maybe embodied in many different forms and should not be construed aslimited to the embodiments set forth herein; rather, these embodimentsare provided so that this disclosure will satisfy applicable legalrequirements. Indeed, many modifications and other embodiments of thepresently disclosed subject matter set forth herein will come to mind toone skilled in the art to which the presently disclosed subject matterpertains having the benefit of the teachings presented in the foregoingdescriptions and the associated Figures. Therefore, it is to beunderstood that the presently disclosed subject matter is not to belimited to the specific embodiments disclosed and that modifications andother embodiments are intended to be included within the scope of theappended claims.

I. PSMA Targeted Fluorescent Agents for Image Guided Surgery

Minimally invasive medical techniques are intended to reduce the amountof extraneous tissue that is damaged during diagnostic or surgicalprocedures, thereby reducing patient recovery time, discomfort, anddeleterious side effects. While millions of “open” or traditionalsurgeries are performed each year in the United States; many of thesesurgeries potentially can be performed in a minimally invasive manner.One effect of minimally invasive surgery, for example, is reducedpost-operative recovery time and related hospital stay. Because theaverage hospital stay for a standard surgery is typically significantlylonger than the average stay for an analogous minimally invasivesurgery, increased use of minimally invasive techniques could savemillions of dollars in hospital costs each year. While many of thesurgeries performed in the United States could potentially be performedin a minimally invasive manner, only a portion currently employ thesetechniques due to instrument limitations, method limitations, and theadditional surgical training involved in mastering the techniques.

Minimally invasive telesurgical systems are being developed to increasea surgeon's dexterity, as well as to allow a surgeon to operate on apatient from a remote location. Telesurgery is a general term forsurgical systems where the surgeon uses some form of remote control,e.g., a servomechanism, or the like, to manipulate surgical instrumentmovements rather than directly holding and moving the instruments byhand. In such a telesurgery system, the surgeon is provided with animage of the surgical site at the remote location. While viewing thesurgical site on a suitable viewer or display, the surgeon performs thesurgical procedures on the patient by manipulating master control inputdevices, which in turn control the motion of instruments. These inputdevices can move the working ends of the surgical instruments withsufficient dexterity to perform quite intricate surgical tasks.

Surgery is the most commonly used treatment for clinically localizedprostate cancer (PCa) and provides a survival advantage compared towatchful waiting. A pressing issue in surgery for PCa is the assuranceof a complete resection of the tumor, namely, a negative surgicalmargin. Surgical techniques, including minimally invasive surgicaltechniques, such as tele-surgical systems, can be further aided byimproving visualization of the tissue where the procedure is to becarried out. One way to improve visualization of tissue is through theuse of dyes capable of targeted visualization of tissue, allowing asurgeon to either remove or spare the tissue.

Accordingly, in some embodiments, the presently disclosed subject matterprovides low-molecular-weight compounds comprising PSMA-targetingligands linked to near-infrared (NIR), closed chain, sulfo-cyanine dyesand methods of their use for visualizing tissue under illumination withNIR radiation, including methods for imaging prostate cancer (PCa).

While a variety of radiolabeled PSMA-targeting antibodies have been usedfor tumor imaging, low molecular weight agents are preferred due to moretractable pharmacokinetics, i.e., more rapid clearance from nontargetsites. A series of fluorescent agents has been previously reported andwas tested in mice to good effect.

See, for example, international PCT patent application publication no.WO2010/108125A2, for PSMA-TARGETING COMPOUNDS AND USES THEREOF, toPomper et al., published Sep. 23, 2010, which is incorporated byreference in its entirety. Because of the favorable pharmacokineticprofile of this class of compounds, i.e., low nonspecific binding, lackof metabolism in vivo and reasonable tumor residence times, this seriesof compounds was extended to include Dylight800 fluorescent dyes. Thus,the presently disclosed compounds include a urea-based PSMA bindingmoiety linked to a Dylight™ 800 fluorescent dye (Thermo FisherScientific Inc., Rockford, Ill., USA). The presently disclosed targetedfluorescent PSMA binding compounds may find utility in fluorescenceimage guided surgery and biopsy of PSMA positive tumors and tissues; theformer providing visual confirmation of complete removal ofPSMA-containing tissue.

A. Compound (3)

Accordingly, in some embodiments, the presently disclosed subject matterprovides the following compound:

or a pharmaceutically acceptable salt thereof.

The presently disclosed compounds can be made using procedures known inthe art by attaching near IR, closed chain, sulfo-cyanine dyes toprostate specific membrane antigen ligands via a linkage. For example,the prostate specific membrane antigen ligands used in the presentlydisclosed compounds can be synthesized as described in international PCTpatent application publication no. WO 2010/108125, to Pomper et al.,published Sep. 23, 2010, which is incorporated herein in its entirety.Compounds can assembled by reactions between different components, toform linkages such as ureas (—NRC(O)NR—), thioureas (—NRC(S)NR—), amides(—C(O)NR— or —NRC(O)—), or esters (—C(O)O— or —OC(O)—). Urea linkagescan be readily prepared by reaction between an amine and an isocyanate,or between an amine and an activated carbonamide (—NRC(O)—). Thioureascan be readily prepared from reaction of an amine with anisothiocyanate. Amides (—C(O)NR— or —NRC(O)—) can be readily prepared byreactions between amines and activated carboxylic acids or esters, suchas an acyl halide or N-hydroxysuccinimide ester. Carboxylic acids mayalso be activated in situ, for example, with a coupling reagent, such asa carbodiimide, or carbonyldiimidazole (CDI). Esters may be formed byreaction between alcohols and activated carboxylic acids. Triazoles arereadily prepared by reaction between an azide and an alkyne, optionallyin the presence of a copper (Cu) catalyst.

Prostate specific membrane antigen ligands can also be prepared bysequentially adding components to a preformed urea, such as thelysine-urea-glutamate compounds described in Banerjee et al. (J.Med.Chem. vol. 51, pp. 4504-4517, 2008). Other urea-based compounds may alsobe used as building blocks.

Exemplary syntheses of the near IR, closed chain, sulfo-cyanine dyesused in the presently disclosed compositions are described in U.S. Pat.Nos. 6,887,854 and 6,159,657 and are incorporated herein in theirentirety. Additionally, some IR, closed chain, sulfo-cyanine dyes of thepresently disclosed subject matter are commercially available, includingDyLight™ 800 (ThermoFisher).

As provided hereinabove, the presently disclosed compounds can besynthesized via attachment of near IR, closed chain, sulfo-cyanine dyesto prostate specific membrane antigen ligands by reacting a reactiveamine on the ligand with a near IR dye. A wide variety of near IR dyesare known in the art, with activated functional groups for reacting withamines.

B. Compositions Comprising Compound (3)

In some embodiments, the presently disclosed subject matter provides acomposition comprising a unit dosage form of compound (3), or apharmaceutically acceptable salt thereof, wherein the composition isadapted for administration to a subject; and wherein, the unit dosageform delivers to the subject an amount between 0.01 mg/kg and 8 mg/kg ofcompound (3). In some embodiments, the composition unit dosage formdelivers to the subject the amount of about 0.01 mg/kg, about 0.05mg/kg, about 0.10 mg/kg, about 0.20 mg/kg, about 0.30 mg/kg, about 0.35mg/kg, about 0.40 mg/kg, about 0.45 mg/kg, about 0.50 mg/kg, about 0.55mg/kg, about 0.60 mg/kg, about 0.65 mg/kg, about 0.70 mg/kg, about 0.75mg/kg, about 0.80 mg/kg, about 0.90 mg/kg, about 1 mg/kg, about 2 mg/kg,about 4 mg/kg, about 6 mg/kg, or about 8 mg/kg. In some embodiments, thecomposition is dry and a single dose form.

The term “unit dosage form” as used herein encompasses any measuredamount that can suitably be used for administering a pharmaceuticalcomposition to a patient. As recognized by those skilled in the art,when another form (e.g., another salt the pharmaceutical composition) isused in the formulation, the weight can be adjusted to provide anequivalent amount of the pharmaceutical composition.

In some embodiments, the composition is lyophilized in a sterilecontainer. In some embodiments, the composition is contained within asterile container, wherein the container has a machine detectableidentifier that is readable by a medical device.

As used herein, the term “sterile” refers to a system or components of asystem free of infectious agents, including, but not limited to,bacteria, viruses, and bioactive RNA or DNA.

As used herein, the term “non-toxic” refers to the non-occurrence ofdetrimental effects when administered to a vertebrate as a result ofusing a pharmaceutical composition at levels effective for visualizationof tissue under illumination with near-infrared radiation (therapeuticlevels).

As used herein, the term “machine detectable identifier” includesidentifiers visible or detectable by machines including medical devices.In some instances, the medical device is a telesurgical system. Machinedetectable identifiers may facilitate the access or utilization ofinformation that is directly encoded in the machine detectableidentifier, or stored elsewhere. Examples of machine detectibleidentifiers include, but are not limited to, microchips, radio frequencyidentification (RFID) tags, barcodes (e.g., 1-dimensional or2-dimensional barcode), data matrices, quick-response (QR) codes, andholograms. One of skill in the art will recognize that other machinedetectible identifiers are useful in the presently disclosed subjectmatter.

In some embodiments, the composition further comprises compound (3) incombination with pharmaceutically acceptable excipients in an oraldosage form. In some embodiments, the composition further comprisescompound (3) in combination with pharmaceutically acceptable carriers inan injectable dosage form. In some embodiments, the composition furthercomprises compound (3) in combination with pharmaceutically acceptableexcipients in a dosage form for direct delivery to a surgical site.

“Pharmaceutically acceptable excipient” and “pharmaceutically acceptablecarrier” refer to a substance that aids the administration of an activeagent to and absorption by a patient and can be included in thepresently disclosed compositions without causing a significant adversetoxicological effect on the patient. Non-limiting examples ofpharmaceutically acceptable excipients include water, NaCl, normalsaline solutions, lactated Ringer's, normal sucrose, normal glucose,binders, fillers, disintegrants, lubricants, coatings, sweeteners,flavors and colors, and the like. One of skill in the art will recognizethat other pharmaceutical excipients are useful in the presentlydisclosed subject matter. Pharmaceutically acceptable carriers includebut not limited to any adjuvants, excipients, glidants, sweeteners,diluents, preservatives, dyes/colorants, flavoring agents, surfactants,wetting agents, dispersing agents, suspending agents, stabilizingagents, isotonic agents, solvents or emulsors.

The term “oral dosage form” as used herein refers to its normal meaningin the art (i.e., a pharmaceutical composition in the form of a tablet,capsule, caplet, gelcap, geltab, pill and the like).

The term “injectable dosage form” as used herein refers to its normalmeaning in the art (i.e., refer to a pharmaceutical composition in theform of solutions, suspensions, and emulsions, for example, water orwater/propylene glycol solutions.) The presently disclosed compositionscan be prepared in a wide variety of oral, parenteral and topical dosageforms. Oral preparations include tablets, pills, powder, dragees,capsules, liquids, lozenges, cachets, gels, syrups, slurries,suspensions, etc., suitable for ingestion by the patient. The presentlydisclosed compositions can also be administered by injection, that is,intravenously, intramuscularly, intracutaneously, subcutaneously,intraduodenally, or intraperitoneally. Also, the compositions describedherein can be administered by inhalation, for example, intranasally.Additionally, the presently disclosed compositions can be administeredtransdermally. The compositions of this invention can also beadministered by intraocular, insufflation, powders, and aerosolformulations (for examples of steroid inhalants, see Rohatagi, J. Clin.Pharmacol. 35:1187-1193, 1995; Tjwa, Ann. Allergy Asthma Immunol.75:107-111, 1995). Accordingly, the presently disclosed subject matteralso provides pharmaceutical compositions including a pharmaceuticallyacceptable carrier or excipient.

For preparing pharmaceutical compositions from the presently disclosedsubject matter, pharmaceutically acceptable carriers can be either solidor liquid. Solid form preparations include powders, tablets, pills,capsules, cachets, suppositories, and dispersible granules. A solidcarrier can be one or more substances, which may also act as diluents,flavoring agents, binders, preservatives, tablet disintegrating agents,or an encapsulating material. Details on techniques for formulation andadministration are well described in the scientific and patentliterature, see, e.g., the latest edition of Remington's PharmaceuticalSciences, Maack Publishing Co, Easton Pa. (“Remington's”).

In powders, the carrier is a finely divided solid, which is in a mixturewith the finely divided active component. In tablets, the activecomponent is mixed with the carrier having the necessary bindingproperties in suitable proportions and compacted in the shape and sizedesired. The powders and tablets preferably contain from 5% or 10% to70% of the compounds of the presently disclosed subject matter.

Suitable solid excipients include, but are not limited to, magnesiumcarbonate; magnesium stearate; talc; pectin; dextrin; starch;tragacanth; a low melting wax; cocoa butter; carbohydrates; sugarsincluding, but not limited to, lactose, sucrose, mannitol, or sorbitol,starch from corn, wheat, rice, potato, or other plants; cellulose suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; and gums including arabic and tragacanth; aswell as proteins including, but not limited to, gelatin and collagen. Ifdesired, disintegrating or solubilizing agents may be added, such as thecross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound (i.e., dosage). Pharmaceutical compositions of theinvention can also be used orally using, for example, push-fit capsulesmade of gelatin, as well as soft, sealed capsules made of gelatin and acoating such as glycerol or sorbitol. Push-fit capsules can contain thepresently disclosed compositions mixed with a filler or binders such aslactose or starches, lubricants such as talc or magnesium stearate, and,optionally, stabilizers. In soft capsules, the presently disclosedcompositions may be dissolved or suspended in suitable liquids, such asfatty oils, liquid paraffin, or liquid polyethylene glycol with orwithout stabilizers.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water/propylene glycol solutions. For parenteralinjection, liquid preparations can be formulated in solution in aqueouspolyethylene glycol solution.

Aqueous solutions suitable for oral use can be prepared by dissolvingthe presently disclosed compositions in water and adding suitablecolorants, flavors, stabilizers, and thickening agents as desired.Aqueous suspensions suitable for oral use can be made by dispersing thefinely divided active component in water with viscous material, such asnatural or synthetic gums, resins, methylcellulose, sodiumcarboxymethylcellulose, hydroxypropylmethylcellulose, sodium alginate,polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing orwetting agents such as a naturally occurring phosphatide (e.g.,lecithin), a condensation product of an alkylene oxide with a fatty acid(e.g., polyoxyethylene stearate), a condensation product of ethyleneoxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partialester derived from a fatty acid and a hexitol (e.g., polyoxyethylenesorbitol mono-oleate), or a condensation product of ethylene oxide witha partial ester derived from fatty acid and a hexitol anhydride (e.g.,polyoxyethylene sorbitan mono-oleate). The aqueous suspension can alsocontain one or more preservatives such as ethyl or n-propylp-hydroxybenzoate, one or more coloring agents, one or more flavoringagents and one or more sweetening agents, such as sucrose, aspartame orsaccharin. Formulations can be adjusted for osmolarity.

Also included are solid form preparations, which are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

Oil suspensions can be formulated by suspending the presently disclosedcompositions in a vegetable oil, such as arachis oil, olive oil, sesameoil or coconut oil, or in a mineral oil such as liquid paraffin; or amixture of these. The oil suspensions can contain a thickening agent,such as beeswax, hard paraffin or cetyl alcohol.

Sweetening agents can be added to provide a palatable oral preparation,such as glycerol, sorbitol or sucrose. These formulations can bepreserved by the addition of an antioxidant such as ascorbic acid. As anexample of an injectable oil vehicle, see Minto, J. Pharmacol. Exp.Ther. 281:93-102, 1997. The pharmaceutical formulations of the inventioncan also be in the form of oil-in-water emulsions. The oily phase can bea vegetable oil or a mineral oil, described above, or a mixture ofthese. Suitable emulsifying agents include naturally-occurring gums,such as gum acacia and gum tragacanth, naturally occurring phosphatides,such as soybean lecithin, esters or partial esters derived from fattyacids and hexitol anhydrides, such as sorbitan mono-oleate, andcondensation products of these partial esters with ethylene oxide, suchas polyoxyethylene sorbitan mono-oleate. The emulsion can also containsweetening agents and flavoring agents, as in the formulation of syrupsand elixirs. Such formulations can also contain a demulcent, apreservative, or a coloring agent.

The presently disclosed compositions can also be delivered asmicrospheres for slow release in the body. For example, microspheres canbe formulated for administration via intradermal injection ofdrug-containing microspheres, which slowly release subcutaneously (seeRao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable andinjectable gel formulations (see, e.g., Gao Pharm. Res. 12:857-863,1995); or, as microspheres for oral administration (see, e.g., Eyles, J.Pharm. Pharmacol. 49:669-674, 1997). Both transdermal and intradermalroutes afford constant delivery for weeks or months.

In another embodiment, the presently disclosed compositions can beformulated for parenteral administration, such as intravenous (IV)administration or administration into a body cavity or lumen of anorgan. The formulations for administration will commonly comprise asolution of the presently disclosed compositions dissolved in apharmaceutically acceptable carrier. Among the acceptable vehicles andsolvents that can be employed are water and Ringer's solution, anisotonic sodium chloride. In addition, sterile fixed oils canconventionally be employed as a solvent or suspending medium. For thispurpose any bland fixed oil can be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid can likewisebe used in the preparation of injectables. These solutions are sterileand generally free of undesirable matter. These formulations may besterilized by conventional, well-known techniques including radiation,chemical, heat/pressure, and filtration sterilization techniques. Theformulations may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents, e.g.,sodium acetate, sodium chloride, potassium chloride, calcium chloride,sodium lactate and the like. The concentration of the presentlydisclosed compositions in these formulations can vary widely, and willbe selected primarily based on fluid volumes, viscosities, body weight,and the like, in accordance with the particular mode of administrationselected and the patient's needs. For IV administration, the formulationcan be a sterile injectable preparation, such as a sterile injectableaqueous or oleaginous suspension. This suspension can be formulatedaccording to the known art using those suitable dispersing or wettingagents and suspending agents. The sterile injectable preparation canalso be a sterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, such as a solution of1,3-butanediol.

In another embodiment, the formulations of the presently disclosedcompositions can be delivered by the use of liposomes which fuse withthe cellular membrane or are endocytosed, i.e., by employing ligandsattached to the liposome, or attached directly to the oligonucleotide,that bind to surface membrane protein receptors of the cell resulting inendocytosis. By using liposomes, particularly where the liposome surfacecarries ligands specific for target cells, or are otherwisepreferentially directed to a specific organ, one can focus the deliveryof the presently disclosed compositions into the target cells in vivo.(See, e.g., Al-Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn,Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm.46:1576-1587, 1989).

Lipid-based drug delivery systems include lipid solutions, lipidemulsions, lipid dispersions, self-emulsifying drug delivery systems(SEDDS) and self-microemulsifying drug delivery systems (SMEDDS). Inparticular, SEDDS and SMEDDS are isotropic mixtures of lipids,surfactants and co-surfactants that can disperse spontaneously inaqueous media and form fine emulsions (SEDDS) or microemulsions(SMEDDS). Lipids useful in the formulations of the presently disclosedsubject matter include any natural or synthetic lipids including, butnot limited to, sesame seed oil, olive oil, castor oil, peanut oil,fatty acid esters, glycerol esters, Labrafil®, Labrasol®, Cremophor®,Solutol®, Tween®, Capryol®, Capmul®, Captex®, and Peceol®.

In some embodiments the presently disclosed compositions are sterile andgenerally free of undesirable matter. The compounds and compositions maybe sterilized by conventional, well known techniques includingheat/pressure, gas plasma, steam, radiation, chemical, and filtrationsterilization techniques.

For example, terminal heat sterilization can be used to destroy allviable microorganisms within the final formulation. An autoclave iscommonly used to accomplish terminal heat-sterilization of drug productsin their final packaging. Typical autoclave cycles in the pharmaceuticalindustry to achieve terminal sterilization of the final product are 121°C. for 15 minutes. The presently disclosed compositions can beautoclaved at a temperature ranging from 115 to 130° C. for a period oftime ranging from 5 to 40 minutes with acceptable stability. Autoclavingis preferably carried out in the temperature range of 119 to 122° C. fora period of time ranging from 10 to 36 minutes.

The compositions can also be sterilized by dry heat as described byKarlsson, et al., in U.S. Pat. No. 6,392,036, which discloses a methodfor the dry heat sterilization that can be used for drug formulations.The compositions can also be sterilized as described in WO 02/41925 toBreath Limited, which discloses a rapid method, similar topasteurization, for the sterilization of compositions. This methodentails pumping the composition to be sterilized through stainless steelpipes and rapidly raising the temperature of the composition to about130-145° C. for about 2-20 seconds, subsequently followed by rapidcooling in seconds to ambient conditions.

The compositions can also be sterilized by irradiation as described byIllum and Moeller in Arch. Pharm. Chem. Sci., Ed. 2, 1974, pp. 167-174).The compositions can also be sterilized by UV, x-rays, gamma rays, ebeam radiation, flaming, baking, and chemical sterilization.

Alternatively, sterile pharmaceutical compositions according to thepresently disclosed subject matter may be prepared using asepticprocessing techniques. Aseptic filling is ordinarily used to preparedrug products that will not withstand heat sterilization, but in whichall of the ingredients are sterile. Sterility is maintained by usingsterile materials and a controlled working environment. All containersand apparatus are sterilized, preferably by heat sterilization, prior tofilling. The container (e.g., vial, ampoule, infusion bag, bottle, orsyringe) are then filled under aseptic conditions.

In some embodiments, the compounds and presently disclosed compositionsare non-toxic and generally free of detrimental effects whenadministered to a vertebrate at levels effective for visualization oftissue under illumination with near-infrared radiation. Toxicity of thecompounds and presently disclosed compositions can be assessed bymeasuring their effects on a target (organism, organ, tissue or cell).Because individual targets typically have different levels of responseto the same dose of a compound, a population-level measure of toxicityis often used which relates the probabilities of an outcome for a givenindividual in a population. Toxicology of compounds can be determined byconventional, well-known techniques including in vitro (outside of aliving organism) and in vivo (inside of a living organism) studies.

For example, determination of metabolic stability is commonly examinedwhen assessing the toxicity of a compound as it is one of several majordeterminates in defining the oral bioavailability and systemic clearanceof a compound. After a compound is administered orally, it firstencounters metabolic enzymes in the gastrointestinal lumen as well as inthe intestinal epithelium. After it is absorbed into the bloodstreamthrough the intestinal epithelium, it is first delivered to the livervia the portal vein. A compound can be effectively cleared by intestinalor hepatic metabolism before it reaches systemic circulation, a processknown as first pass metabolism. The stability of a compound towardsmetabolism within the liver as well as extrahepatic tissues willultimately determine the concentration of the compound found in thesystemic circulation and affect its half-life and residence time withinthe body. Cytochrome P450 (CYP) enzymes are found primarily in the liverbut also in the intestinal wall, lungs, kidneys and other extrahepaticorgans and are the major enzymes involved in compound metabolism. Manycompounds undergo deactivation by CYPs, either directly or byfacilitated excretion from the body. Also, many compounds arebioactivated by CYPs to form their active compounds. Thus, determiningthe reactivity of a compound to CYP enzymes is commonly used to assessmetabolic stability of a compound.

The Ames reverse mutation Assay is another common toxicology assay forassessing the toxicity of a compound. The Ames Assay, utilizes severaldifferent tester strains, each with a distinct mutation in one of thegenes comprising the histidine (his) biosynthetic operon (Ames, B. N.,et al., (1975) Mutation Res. 31:347-363). The detection of revertant(i.e., mutant) bacteria in test samples that are capable of growth inthe absence of histidine indicates that the compound under evaluation ischaracterized by genotoxic (i.e. mutagenic) activity. The Ames Assay iscapable of detecting several different types of mutations (geneticdamage) that may occur in one or more of the tester strains. Thepractice of using an in vitro bacterial assay to evaluate the genotoxicactivity of drug candidates is based on the prediction that a substancethat is mutagenic in a bacterium is likely to be carcinogenic inlaboratory animals, and by extension may be carcinogenic or mutagenic tohumans.

In addition, the human ether-a-go-go related gene (hERG) assay can beused to evaluate the potential cardiotoxicity of a compound.Cardiotoxicity can arise when the QT interval is prolonged leading to anelevated risk of life-threatening arrhythmias. The QT interval is ameasure of the time between the start of the Q wave and the end of the Twave in the heart's electrical cycle. The QT interval representselectrical depolarization and repolarization of the ventricles. Alengthened QT interval has most commonly been associated with loss ofcurrent through hERG potassium ion channels due to direct block of theion channel by drugs or by inhibition of the plasma membrane expressionof the channel protein (Su et al. J. Biomol Screen 2011, 16, 101-111).Thus, an in vitro hERG screening assay can be used to detect disruptionor inhibition of the hERG membrane trafficking function and assesspotential cardiotoxicity of a compound.

Other methods of assessing the toxicity of compounds include in vivostudies which administer relatively large doses of a test compound to agroup of animals to determine the level which is lethal to a percentageof the population (mean lethal dose LD₅₀ or mean lethal concentrationLC₅₀). Toxicity of a compound can also be assessed in vivo by examiningwhether a compound produces statistically significant negative effectson cardiac, blood pressure, central nervous system (CNS), body weight,food intake, gross or microscopic pathology, clinical pathology, orrespiratory measures in an animal.

In some embodiments, the presently disclosed compositions can belyophilized in a sterile container for convenient dry storage andtransport. A ready-to-use preparation can subsequently be made byreconstituting the lyophilized compositions with sterile water. Theterms “lyophilization,” “lyophilized,” and “freeze-dried” refer to aprocess by which the material to be dried is first frozen and then theice or frozen solvent is removed by sublimation in a vacuum environment.An excipient may be included in pre-lyophilized formulations to enhancestability of the lyophilized product upon storage.

In some embodiments, the composition can be contained within a sterilecontainer, where the container has a machine detectable identifier whichis readable by a medical device. Examples of machine detectibleidentifiers include microchips, radio frequency identification (RFID)tags, barcodes (e.g., 1-dimensional or 2-dimensional barcode), datamatrices, quick-response (QR) codes, and holograms. One of skill in theart will recognize that other machine detectible identifiers are usefulin the presently disclosed subject matter.

In some embodiments, the machine detectable identifier can include amicrochip, an integrated circuit (IC) chip, or an electronic signal froma microchip that is detectable and/or readable by a computer system thatis in communication with the medical device. In some embodiments, themachine detectable identifier includes a radio frequency identification(RFID) tag. RFID tags are sometimes called as transponders. RFID tagsgenerally are devices formed of an IC chip, an antenna, an adhesivematerial, and are used for transmitting or receiving predetermined datawith an external reader or interrogator. RFID tags can transmit orreceive data with a reader by using a contactless method. According tothe amplitude of a used frequency, inductive coupling, backscattering,and surface acoustic wave (SAW) may be used.

Using electromagnetic waves, data may be transmitted or received to orfrom a reader by using a full duplex method, a half duplex (HDX) method,or a sequential (SEQ) method.

In some embodiments, the machine detectable identifier can include abarcode. Barcodes include any machine-readable format, includingone-dimensional and two-dimensional formats. One-dimensional formatsinclude, for example, Universal Product Code (UPC) and Reduced SpaceSymbology (RSS). Two-dimensional formats, or machine-readable matrices,include for example, Quick Response (QR) Code and Data Matrix.

In some embodiments, the medical device can be configured to detect themachine detectable identifier. In one example, the medical device is atele-surgical system that includes a special imaging mode (e.g., afluorescence imaging mode) for use with dyes such as those described inthis disclosure. One example of a tele-surgical system that includes afluorescence imaging mode is described in U.S. Pat. No. 8,169,468,entitled “Augmented Stereoscopic Visualization for a Surgical Robot,”which is hereby incorporated in its entirety herein. In someembodiments, medical devices can incorporate an imaging device that canscan, read, view, or otherwise detect a machine detectable identifierthat is displayed to the imaging device. In one aspect, the medicaldevice will permit a user to access the fluorescence imaging mode of themedical device only if the medical device detects the presence of aknown machine detectable identifier that corresponds to a dye identifiedas being compatible for use with the medical device. In contrast, if themedical device does not detect a known machine detectable identifier,the medical device will not permit a user to access the fluorescenceimaging mode and associated functionality. Imaging devices can includeoptical scanners, barcode readers, cameras, and imaging devicescontained within a tele-surgical system such as an endoscope.Information associated with the machine detectable identifier can thenbe retrieved by the medical device using an imaging device. Upondetection of the identifier, an automatic process may be launched tocause a predetermined action to occur, or certain data to be retrievedor accessed. The information encoded onto the machine detectableidentifier may include instructions for triggering an action, such asadministering a composition of the presently disclosed subject matter toa patient. In some embodiments, the machine detectable identifierincludes unencrypted e-pedigree information in the desired format. Thee-pedigree information can include, for example, lot, potency,expiration, national drug code, electronic product code, manufacturer,distributor, wholesaler, pharmacy and/or a unique identifier of thesalable unit.

In some embodiments, the sterile container having a machine detectableidentifier includes a fluid outlet configured to mate with the medicaldevice. In some embodiments, the fluid outlet of the machine detectableidentifier is mechanically affixed to the medical device.

C. Methods of Imaging Using Compositions Comprising Compound (3)

In some embodiments, the presently disclosed subject matter provides ause of the composition comprising compound (3), or a pharmaceuticallyacceptable salt thereof, adapted for administration to a subject, e.g.,a patient, to obtain visualization of tissue expressing PSMA underillumination with near-infrared radiation, wherein the unit dosage formdelivers to the subject an amount between about 0.01 mg/kg and 8 mg/kgof compound (3). In some embodiments, the use is adapted foradministration to a human patient to obtain visualization of humantissue under illumination with near-infrared radiation wherein the unitdosage form delivers to the human patient an amount between about 0.01mg/kg and 8 mg/kg of a compound (3).

The compounds and presently disclosed compositions can be delivered byany suitable means, including oral, parenteral and topical methods.Transdermal administration methods, by a topical route, can beformulated as applicator sticks, solutions, suspensions, emulsions,gels, creams, ointments, pastes, jellies, paints, powders, and aerosol.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the compounds and presently disclosedcompositions. The unit dosage form can be a packaged preparation, thepackage containing discrete quantities of preparation, such as packetedtablets, capsules, and powders in vials or ampoules. Also, the unitdosage form can be a capsule, tablet, cachet, or lozenge itself, or itcan be the appropriate number of any of these in packaged form.

In some embodiments, co-administration can be accomplished byco-formulation, i.e., preparing a single pharmaceutical compositionincluding the compounds and presently disclosed compositions and anyother agent. Alternatively, the various components can be formulatedseparately.

The presently disclosed compositions, and any other agents, can bepresent in any suitable amount, and can depend on various factorsincluding, but not limited to, weight and age of the patient, state ofthe disease, etc. Suitable dosage ranges include from about 0.01 and 8mg/kg, or about 0.01 and 5 mg/kg, or about 0.01 and 1 mg/kg. Suitabledosage ranges also include 0.01, 0.05, 0.10, 0.20, 0.30, 0.35, 0.40,0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.90, 1, 2, 4, 6, or 8mg/kg.

The composition can also contain other compatible compositions. Thecompositions described herein can be used in combination with oneanother, with other active compositions known to be useful forvisualization of tissue under illumination with near-infrared radiation,or with compositions that may not be effective alone, but may contributeto the efficacy of the active composition.

As used herein, the term “visualization” refers to methods of obtaininggraphic images of tissue by any means, including illumination withnear-infrared radiation.

The term “near-infrared radiation” or “near IR radiation” or “NIR”radiation refers to optical radiation with a wavelength in the range ofabout 700 nm to about 1400 nm. References herein to the optionallyplural term “wavelength(s)” indicates that the radiation may be a singlewavelength or a spectrum of radiation having differing wavelengths.

The term “tissue” as used herein includes, but is not limited to,allogenic or xenogenic bone, neural tissue, fibrous connective tissueincluding tendons and ligaments, cartilage, dura, fascia, pericardia,muscle, heart valves, veins and arteries and other vessels, dermis,adipose tissue, glandular tissue, prostate tissue, kidney tissue, braintissue, renal tissue, bladder tissue, lung tissue, breast tissue,pancreatic tissue, vascular tissue, tumor tissue, cancerous tissue, orprostate tumor tissue.

In particular embodiments, the presently disclosed subject matterprovides a method for visualization of tissue expressing PSMA, themethod comprising, administering to a subject, e.g., a patient, acomposition comprising compound (3), described herein. In someembodiments, the method comprises, administering to a subject acomposition comprising compound (3):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the method administers to a subject apharmaceutical composition comprising a unit dosage form of compound(3), wherein the composition is sterile, non-toxic, and adapted foradministration to a subject; and wherein, the unit dosage form deliversto the subject an amount between about 0.01 mg/kg and 8 mg/kg ofcompound (3). In some embodiments, the method further comprisesobtaining the image during administration, after administration, or bothduring and after administration of the composition. In some embodiments,the method further comprises intravenously injecting a compositioncomprising compound (3) into a subject. In some embodiments, thecomposition is injected into a circulatory system of the subject.

In some embodiments, the method further comprises visualizing a subjectarea on which surgery is or will be performed, or for viewing a subjectarea otherwise being examined by a medical professional. In someembodiments, the method further comprises performing a surgicalprocedure on the subject areas based on the visualization of thesurgical area. In some embodiments, the method further comprises viewinga subject area on which an ophthalmic, arthroscopic, laparoscopic,cardiothoracic, muscular, or neurological procedure is or will beperformed. In some embodiments, the method further comprises obtainingex vivo images of at least a portion of the subject.

In some embodiments, the tissue being visualized is tumor tissue. Insome embodiments, the tissue being visualized is dysplastic or canceroustissue. In some embodiments, the tissue being visualized is prostatetissue. In some embodiments, the tissue being visualized is prostatetumor tissue.

In other embodiments, the one or more PSMA-expressing tumor or cell isselected from the group consisting of: a prostate tumor or cell, ametastasized prostate tumor or cell, a lung tumor or cell, a renal tumoror cell, a glioblastoma, a pancreatic tumor or cell, a bladder tumor orcell, a sarcoma, a melanoma, a breast tumor or cell, a colon tumor orcell, a germ cell, a pheochromocytoma, an esophageal tumor or cell, astomach tumor or cell, and combinations thereof. In more particularembodiments, the one or more PSMA-expressing tumor or cell is a prostatetumor or cell. In certain embodiments, the one or more PSMA-expressingtumors or cells are in vitro, in vivo, or ex vivo. In particularembodiments, the one or more PSMA-expressing tumors or cells are presentin a subject.

The “subject” treated by the presently disclosed methods in their manyembodiments is desirably a human subject, although it is to beunderstood that the methods described herein are effective with respectto all vertebrate species, which are intended to be included in the term“subject.” Accordingly, a “subject” can include a human subject formedical purposes, such as for the treatment of an existing condition ordisease or the prophylactic treatment for preventing the onset of acondition or disease, or an animal subject for medical, veterinarypurposes, or developmental purposes. Suitable animal subjects includemammals including, but not limited to, primates, e.g., humans, monkeys,apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines,e.g., sheep and the like; caprines, e.g., goats and the like; porcines,e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras,and the like; felines, including wild and domestic cats; canines,including dogs; lagomorphs, including rabbits, hares, and the like; androdents, including mice, rats, and the like. An animal may be atransgenic animal. In some embodiments, the subject is a humanincluding, but not limited to, fetal, neonatal, infant, juvenile, andadult subjects. Further, a “subject” can include a patient afflictedwith or suspected of being afflicted with a condition or disease. Thus,the terms “subject” and “patient” are used interchangeably herein. Theterm “subject” also refers to an organism, tissue, cell, or collectionof cells from a subject.

In general, the “effective amount” of an active agent refers to theamount necessary to elicit the desired biological response. As will beappreciated by those of ordinary skill in this art, the effective amountof an agent or device may vary depending on such factors as the desiredbiological endpoint, the agent to be delivered, the makeup of thepharmaceutical composition, the target tissue, and the like.

In some embodiments, compound (3) is cleared from the subject's kidneysin about 24 hours.

In some embodiments, the presently disclosed methods use compounds thatare stable in vivo such that substantially all, e.g., more than about50%, 60%, 70%, 80%, or more preferably 90% of the injected compound isnot metabolized by the body prior to excretion. In other embodiments,the compound comprising the imaging agent is stable in vivo.

D. PSMA-Targeting Compounds and Uses Thereof

In further embodiments, the presently disclosed subject matter providesa compound having the structure:

wherein: Z is tetrazole or CO₂Q; each Q is independently selected fromhydrogen or a protecting group; FG is a fluorescent dye moiety whichemits in the visible or near infrared spectrum; each R is independentlyH or C₁-C₄ alkyl; V is —C(O)—; W is —NRC(O); Y is —C(O); a is 1, 2, 3,or 4; m is 1, 2, 3, 4, 5, or 6; n is 1, 2, 3, 4, 5 or 6; p is 0, 1, 2,or 3, and when p is 2 or 3, each R¹ may be the same or different; R¹ isH, C₁-C₆ alkyl, C₆-C₁₂ aryl, or alkylaryl having 1 to 3 separate orfused rings and from 6 to about 18 ring carbon atoms; R² and R³ areindependently H, CO₂H, or CO₂R⁴, where R⁴ is a C₁-C₆ alkyl, C₆-C₁₂ aryl,or alkylaryl having 1 to 3 separate or fused rings and from 6 to about18 ring carbon atoms, wherein when one of R² and R³ is CO₂H or CO₂R⁴,the other is H.

As used herein, a “protecting group” is a chemical substituent which canbe selectively removed by readily available reagents which do not attackthe regenerated functional group or other functional groups in themolecule. Suitable protecting groups are known in the art and continueto be developed. Suitable protecting groups may be found, for example inWutz et al. (“Greene's Protective Groups in Organic Synthesis, FourthEdition,” Wiley-Interscience, 2007). Protecting groups for protection ofthe carboxyl group, as described by Wutz et al. (pages 533-643), areused in certain embodiments. In some embodiments, the protecting groupis removable by treatment with acid. Specific examples of protectinggroups include but are not limited to, benzyl, p-methoxybenzyl (PMB),tertiary butyl (t-Bu), methoxymethyl (MOM), methoxyethoxymethyl (MEM),methylthiomethyl (MTM), tetrahydropyranyl (THP), tetrahydrofuranyl(THF), benzyloxymethyl (BOM), trimethylsilyl (TMS), triethylsilyl (TES),t-butyldimethylsilyl (TBDMS), and triphenylmethyl (trityl, Tr). Personsskilled in the art will recognize appropriate situations in whichprotecting groups are required and will be able to select an appropriateprotecting group for use in a particular circumstance.

In certain embodiments, the compound has the following structure:

In more certain embodiments, the compound has the following structure:

In yet more certain embodiments, the compound has the followingstructure:

In particular embodiments, R³ is CO₂H and R² is H or R² is CO₂H and R³is H. In other embodiments, R² is CO₂R⁴ and R³ is H or R³ is CO₂R⁴, andR² is H. In yet other embodiments, R² is H, and R³ is H.

In certain embodiments, R⁴ is C₆-C₁₂ aryl, or alkylaryl having 1 to 3separate or fused rings and from 6 to about 18 ring carbon atoms. Incertain embodiments, R¹ is C₆-C₁₂ aryl. In more certain embodiments, R¹is phenyl.

In particular embodiments, FG is a fluorescent dye moiety which emits inthe near infrared spectrum. In more particular embodiments, FG comprisesa fluorescent dye moiety selected from the group consisting ofcarbocyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine andmerocyanine, polymethine, coumarine, rhodamine, xanthene, fluorescein,boron-dipyrromethane (BODIPY), Cy5, Cy5.5, Cy7, VivoTag-680,VivoTag-S680, VivoTag-S750, AlexaFluor660, AlexaFluor680, AlexaFluor700,AlexaFluor750, AlexaFluor790, Dy677, Dy676, Dy682, Dy752, Dy780,DyLight547, Dylight647, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor750, IRDye 800CW, IRDye 800RS, IRDye 700DX, ADS780WS, ADS830WS, andADS832WS. In yet more particular embodiments, FG has a structureselected from the group consisting of:

In some embodiments, the compound is selected from the group consistingof:

E. Imaging

Embodiments include methods of imaging one or more cells, organs ortissues comprising exposing cells to or administering to a subject aneffective amount of a compound with an isotopic label suitable forimaging. In some embodiments, the one or more organs or tissues includeprostate tissue, kidney tissue, brain tissue, vascular tissue or tumortissue. The cells, organs or tissues may be imaged while within anorganism, either by whole body imaging or intraoperative imaging, or maybe excised from the organism for imaging.

In another embodiment, the imaging method is suitable for imagingstudies of PSMA inhibitors, for example, by studying competitive bindingof non-radiolabeled inhibitors. In still another embodiment, the imagingmethod is suitable for imaging of cancer, tumor or neoplasm. In afurther embodiment, the cancer is selected from eye or ocular cancer,rectal cancer, colon cancer, cervical cancer, prostate cancer, breastcancer and bladder cancer, oral cancer, benign and malignant tumors,stomach cancer, liver cancer, pancreatic cancer, lung cancer, corpusuteri, ovary cancer, prostate cancer, testicular cancer, renal cancer,brain cancer (e.g., gliomas), throat cancer, skin melanoma, acutelymphocytic leukemia, acute myelogenous leukemia, Ewing's Sarcoma,Kaposi's Sarcoma, basal cell carcinoma and squamous cell carcinoma,small cell lung cancer, choriocarcinoma, rhabdomyosarcoma, angiosarcoma,hemangioendothelioma, Wilms Tumor, neuroblastoma, mouth/pharynx cancer,esophageal cancer, larynx cancer, lymphoma, neurofibromatosis, tuberoussclerosis, hemangiomas, and lymphangiogenesis.

The imaging methods of the invention are suitable for imaging anyphysiological process or feature in which PSMA is involved. Typically,imaging methods are suitable for identification of areas of tissues ortargets which express high concentrations of PSMA. Typical applicationsinclude imaging glutamateric neurotransmission, presynapticglutamatergic neurotransmission, malignant tumors or cancers thatexpress PSMA, prostate cancer (including metastasized prostate cancer),and angiogenesis. Essentially all solid tumors express PSMA in theneovasculture. Therefore, methods of the present invention can be usedto image nearly all solid tumors including lung, renal cell,glioblastoma, pancreas, bladder, sarcoma, melanoma, breast, colon, germcell, pheochromocytoma, esophageal and stomach. Also, certain benignlesions and tissues including endometrium, schwannoma and Barrett'sesophagus can be imaged according to the present invention.

The methods of imaging angiogenesis are suitable for use in imaging avariety of diseases and disorders in which angiogenesis takes place.Illustrative, non-limiting, examples include tumors, collagen vasculardisease, cancer, stroke, vascular malformations, and retinopathy.Methods of imaging angiogenesis are also suitable for use in diagnosisand observation of normal tissue development.

PSMA is frequently expressed in endothelial cells of capillary vesselsin peritumoral and endotumoral areas of various malignancies such thatcompounds of the invention and methods of imaging using same aresuitable for imaging such malignancies.

Imaging agents of the invention may be used in accordance with themethods of the invention by one of skill in the art. Images can begenerated by virtue of differences in the spatial distribution of theimaging agents which accumulate at a site when contacted with PSMA. Thespatial distribution may be measured using any means suitable for theparticular label, for example, a fluorescence camera and the like.

In general, a detectably effective amount of the imaging agent isadministered to a subject. As used herein, “a detectably effectiveamount” of the imaging agent is defined as an amount sufficient to yieldan acceptable image using equipment which is available for clinical use.A detectably effective amount of the imaging agent may be administeredin more than one injection. The detectably effective amount of theimaging agent can vary according to factors such as the degree ofsusceptibility of the individual, the age, sex, and weight of theindividual, idiosyncratic responses of the individual, and thedosimetry. Detectably effective amounts of the imaging agent can alsovary according to instrument and film-related factors. Optimization ofsuch factors is well within the level of skill in the art. The amount ofimaging agent used for diagnostic purposes and the duration of theimaging study will depend upon the radionuclide used to label the agent,the body mass of the patient, the nature and severity of the conditionbeing treated, the nature of therapeutic treatments which the patienthas undergone, and on the idiosyncratic responses of the patient.Ultimately, the attending physician will decide the amount of imagingagent to administer to each individual patient and the duration of theimaging study.

In some embodiments, the compounds are excreted from tissues of the bodyquickly. Generally, the compounds are excreted from tissues of the bodyslowly enough to allow sufficient time for imaging or other use.Typically compounds of the invention are eliminated from the body inless than about 24 hours. More typically, compounds of the invention areeliminated from the body in less than about 16 hours, 12 hours, 8 hours,6 hours, 4 hours, 2 hours, 90 minutes, or 60 minutes. Exemplarycompounds are eliminated in between about 60 minutes and about 120minutes.

In some embodiments of the invention, the compounds are designed toincrease uptake in PSMA positive cells (i.e., tumor cells). For example,highly hydrophilic compounds may be excreted quickly. Compounds withincreased hydrophobicity, such as compounds having hydrophobic linkers,may have longer circulation times, thereby providing more prolongedsupply of tracer to bind to cells. According to embodiments of compoundsaccording to the invention, hydrophobicity can be increased when, forexample, p is 1 or more, or when R² or R³ is CO₂R⁴.

Accordingly, in some embodiments, the presently disclosed subject matterprovides a method of imaging one or more cells, organs or tissues byexposing the cell to or administering to an organism an effective amountof a presently disclosed compound, where the compound includes afluorescent dye moiety suitable for imaging.

F. Cell Sorting

Embodiments include methods for sorting cells by exposing the cells to acompound discussed above, where the compound includes a fluorescent dyemoiety, followed by separating cells which bind the compound from cellswhich do not bind the compound.

Fluorescent compounds described above bind to PSMA on cells that expressPSMA on the cell surface. In some cases, fluorescent compound isinternalized. Cells binding the fluorescent compound appear fluorescent,and may be imaged using fluorescence microscopy. Fluorescence-activatedcell sorting (FACS) or flow cytometry may be used to separate PSMApositive cells from PSMA negative cells.

Intraoperative Tumor Mapping

Embodiments of the invention include methods of intraoperative tumormapping or intraoperative photodiagnosis (PDD) by administering aneffective amount of a compound discussed above to a subject, where thecompound includes a fluorescent dye moiety. According to suchembodiments, an effective amount of a compound is an amount sufficientto produce a detectable level of fluorescence when used forintraoperative tumor mapping or PDD. The compounds bind to, and may beinternalized into, cells, particularly tumor cells, that express PSMA.The fluorescent compounds thereby define the boundaries of the tumor,allowing for accurate surgical removal. The compounds that includes afluorescent dye moiety may also be used to visualize circulating tumorcells that express PSMA.

Kits

Other embodiments provide kits including a compound according to theinvention. In certain embodiments, the kit provides packagedpharmaceutical compositions having a pharmaceutically acceptable carrierand a compound of the invention. In some embodiments the packagedpharmaceutical composition will include the reaction precursorsnecessary to generate the compound of the invention. Other packagedpharmaceutical compositions provided by the present invention furthercomprise indicia comprising at least one of: instructions for preparingcompounds according to the invention from supplied precursors,instructions for using the composition to image cells or tissuesexpressing PSMA, or instructions for using the composition to imageglutamatergic neurotransmission in a patient suffering from astress-related disorder, or instructions for using the composition toimage prostate cancer.

The imaging agent and carrier may be provided in solution or inlyophilized form. When the imaging agent and carrier of the kit are inlyophilized form, the kit may optionally contain a sterile andphysiologically acceptable reconstitution medium such as water, saline,buffered saline, and the like. The kit may provide a compound of theinvention in solution or in lyophilized form, and these components ofthe kit of the invention may optionally contain stabilizers such asNaCl, silicate, phosphate buffers, ascorbic acid, gentisic acid, and thelike. Additional stabilization of kit components may be provided in thisembodiment, for example, by providing the reducing agent in anoxidation-resistant form. Determination and optimization of suchstabilizers and stabilization methods are well within the level of skillin the art.

G. PMA-Targeting Compounds Comprising Metal Chelating Moieties and UsesThereof G.1 Embodiments

As described herein, all embodiments or subcombinations may be used incombination with all other embodiments or subcombinations, unlessmutually exclusive.

In some of the following embodiments, Z is CO₂Q. In some of thefollowing embodiments, Q is H. In some of the following embodiments. mis 4, 5. or 6. In some of the following embodiments. m is 6. In some ofthe following embodiments, n is 2, 3, or 4. In some of the followingembodiments, n is 3. In some of the following embodiments, a is 3 or 4.In some of the following embodiments, a is 4. In some of the followingembodiments, Y is —C(O)—. In some of the following embodiments, W is—NHC(O)—.

Embodiments of the invention include compounds having the structure

wherein the subunits associated with elements p, q, r, and s may be inany order. Z is tetrazole or CO₂Q; each Q is independently selected fromhydrogen or a protecting group, a is 1, 2, 3, or 4, and R is eachindependently H or C₁-C₄ alkyl.

Variable r is 0 or 1. Tz is a triazole group selected from the groupconsisting of

where L¹ is

L² is f-(CH₂)_(b)— — or

X¹ is —NRC(O)—, —NRC(O)NR—, —NRC(S)NR—, or —NRC(O)O—;

X² is-C(O)NR—, —NRC(O)NR—, —NRC(S)NR—, Or —OC(O)NR—;

R⁵ is H, CO₂H, or CO₂R, where R⁶ is a C₁-C₆ alkyl, C₂-C₁₂ aryl, orC₄-C₁₆ alkylaryl;

b is 1, 2, 3, or 4; and d is 1, 2, 3, or 4.

Variable q is 0 or 1. W is —NRC(O)—, —NRC(O)NR—, NRC(S)NR—, —NRC(O)O—,—OC(O)NR—, —OC(O)—, —C(O)NR—, or —C(O)O—; R² and R³ are independently H,CO₂H, or CO₂R⁴, where R⁴ is a C₁-C₆ alkyl, C₂-C₁₂ aryl, or C₄-C₁₆alkylaryl, wherein if one of R² and R³ is CO₂H or CO₂R², then the otheris H;

n is 1, 2, 3, 4, 5 or 6.

Variables is O or 1.

Y is —C(O)—, —NRC(O)—, —NRC(S)—, —OC(O)—; and

m is 1, 2, 3, 4, 5, or 6.

Variable p is 0, 1, 2, or 3, and when p is 2 or 3, each R¹ may be thesame or different.

R¹ is H, C₁-C₆ alkyl, C₂-C₁₂ aryl, or C₄-C₁₆ alkylaryl.

G is a moiety selected from the group consisting of

where Ch is a metal chelating moiety, optionally including a chelatedmetal; FG is a fluorescent dye moiety which emits in the visible or nearinfrared spectrum; one of A and A¹ is Ch and the other is FG;

V and V′ are independently —C(O)—, —NRC(O)—, —NRC(S)—, or —OC(O)—; and

g is 1, 2, 3, 4, 5. or 6.

The following conditions also apply:

1) when G is

and r is 0, then q and s are both 1;2) when G is

and r is 0, then q and s are both 0 or both 1;3) when G is

then p is 0 and R2 is H, and the structure optionally includes achelated metal ion;4) when G is

and r is 0, then if p is 0, then one of R² and R³ is CO₂R², and theother is H; and5) when g is

then r is 0.

In some embodiments. Z is CO₂Q. In some embodiments, Q is H. In someembodiments, m is 4, 5, or 6. In some embodiments, m is 6. In someembodiments, n is 2, 3, or 4. In some embodiments, n is 3. In someembodiments, a is 4. In some embodiments, subunits associated withelements p, q and s are in the order drawn and r may be in any location,including between one of p, q, or s. In some embodiments r is 0.

Embodiments include compounds having the structure

wherein

Z is tetrazole or CO₂Q;

each Q is independently selected from hydrogen or a protecting group,

a is 1, 2, 3, or 4, and R is each independently H or C₁-C₄ alkyl.

Ch is a metal chelating moiety optionally including a chelated metal.

W is —NRC(O)—, —NRC(O)NR—, NRC(S)NR—, —NRC(O)O—, —OC(O)NR—, —OC(O)—,—C(O)NR—, or —C(O)O—.

Y is —C(O)—, —NRC(O)—, —NRC(S)—, —OC(O).

V is —C(O)—, —NRC(O)—, —NRC(S)—, or —OC(O)—.

In exemplary embodiments:

m is 1, 2, 3, 4, 5, or 6:

n is 1, 2, 3, 4, 5 or 6; and

p is 0, 1, 2, or 3, and when p is 2 or 3, each R¹ may be the same ordifferent.

R is H. C₁-C₆ alkyl, C₂-C₁₂ aryl or C₄-C₁₆ alkylaryl.

R and R are independently H, CO₂H, or CO₂R⁴, where R⁴ is a C₁-C₆ alkyl,C₂-C₁₂ aryl, or C₄-C₁₆ alkylaryl, wherein when one of R² and R³ is CO₂Hor CO₂R², the other is H, and when p is 0, one of R² and R³ is CO₂R⁴,and the other is H.

In some embodiments, the compound has the structure shown below.

In some embodiments, the compound has the structure shown below.

In some embodiments, p is 1, 2 or 3. When p is 2 or 3, each R¹ may bethe same or different. When two R¹ groups are different, the two may bein any order. In some embodiments, p is 2. In some embodiments, p is 2,and both R¹ are the same. In some embodiments, R¹ is C₂-C₁₂ aryl. Insome embodiments R¹ is phenyl. In some embodiments. R³ is CO₂H and R² isH. In some embodiments, R² is CO₂H and R³ is H. In some embodiments, R²and R³ are both H.

In some embodiments, p is 0. In some embodiments where p is 0, R² isCO₂R⁴, and R³ is H. In some embodiments where p is 0. R³ is CO₂R⁴, andR² is H. In some embodiments R⁴ is C₂-C₁₂ aryl, or C₄-C₁₆ alkylaryl. Insome embodiments R⁴ is benzyl.

Ch is a metal chelating moiety optionally including a chelated metal. Ametal chelating moiety is a chemical moiety that non-covalently binds ametal atom, usually with a plurality of non-covalent interactions. Chincludes any additional atoms or linkers necessary to attach the metalchelating moiety to the rest of the compound. For instance linkinggroups having alkyl, aryl, combination of alkyl and aryl, or alkyl andaryl groups having heteroatoms may be present in the chelating moiety.Numerous metal chelating moieties are known in the art. Any acceptablechelator can be used with the present invention as long as compatibleand capable of chelating a desired metal. Examples of metal chelatingmoieties (Ch) include, but are not limited to1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) andDiethylene-triaminepentaacetic acid (DTPA).

In some embodiments, Ch has a structure shown below.

Examples of specific compounds include the compounds shown below.

In some embodiments, the compound further includes a chelated metal. Insome embodiments, the chelated metal is Tc, In, Ga, Y, Lu, Re, Cu, Ac,Bi, Pb, Sm, Sc, Co, Ho, Gd, Eu, Tb, or Dy. In some embodiments, thechelated metal is Tc, Ga, In, Cu. Y, Ac, Lu, Re, or Bi. In someembodiments the metal is an isotope, for example a radioactive isotope.In some embodiments, the isotope is Tc-99m, In-111, Ga-67, Ga-68, Y-86,Y-90, Lu-177, Re-186, Re-188, Cu-64, Cu-67, Co-55, Co-57, Sc-47, Ac-225,Bi-213, Bi-212, Pb-212, Sm-153, Ho-166, or Dy-166. In some embodiments,the isotope is Tc-99m. In-111, Ga-67, Ga-68, Y-90, Lu-177, Re-186,Re-188, Cu-67, Ac-225, Bi-213 or Bi-212.

Accordingly, in some embodiments, the presently disclosed subject matterprovides a compound having the structure:

wherein:

Z is tetrazole or CO₂Q;

each Qis independently selected from hydrogen or a protecting group;

a is 1, 2, 3, or 4;

R is each independently H or C₁-C₄ alkyl:

Ch is a metal chelating moiety optionally including a chelated metal,wherein Ch optionally includes any additional atoms or linkers necessaryto attach the metal chelating moiety to the rest of the compound;

W is —NRC(O)—, —NRC(O)NR—, NRC(S)NR—, —NRC(O)O—, —OC(O)NR—, —OC(O)—,—C(O)NR—, or —C(O)O—;

Y is —C(O)—, —NRC(O)—, —NRC(S)—, —OC(O);

V is —C(O)—, —NRC(O)—, —NRC(S)—, or —OC(O)—;

m is 1, 2, 3, 4, 5, or 6;

n is 1, 2, 3, 4, 5 or 6;

p is 0, 1, 2, or 3, and when p is 2 or 3, each R may be the same ordifferent;

R¹ is H, C₁-C₆ alkyl, C₂-C₁₂ aryl, or C₄-C₁₆ alkylaryl;

R² and R³ are independently H, CO₂H, or CO₂R⁴, wherein R⁴ is a C₁-C₆alkyl, C₂-C₁₂ aryl, or C₄-C₁₆ alkylaryl, wherein when one of R² and R³is CO₂H or CO₂R⁴, the other is H, and when p is 0, one of R² and R³ isCO₂R⁴, and the other is H; and

pharmaceutically acceptable salts thereof.

In some embodiments, the compound of formula (I) has the structure:

In some embodiments of the compound of formula (I), Z is CO₂Q; each Q ishydrogen; R is H; a is 4; m is 6; n is 3; p is 2; R¹ is C₂-C₁₂ aryl,wherein the aryl may be substituted or unsubstituted and R¹ may be thesame or different; R² is CO₂H; R³ is H; W is —NRC(O)—, wherein R is H; Vis —C(O)—; and Ch includes any additional atoms or linkers necessary toattach the metal chelating moiety to the rest of the compound.

In certain embodiments, R¹ is phenyl or a substituted phenyl. Inparticular embodiments, R¹ is a phenyl substituted at 1, 2, 3, or 4positions with a substituent group selected from the group consisting ofhalogen, cyano, hydroxyl, nitro, azido, amino, alkanoyl, carboxamido,alkyl, alkenyl, alkynyl, alkoxy, aryloxy, alkylthio, alkylsulfinyl,alkylsulfonyl, aminoalkyl, carbocyclic aryl, arylalkyl, arylalkoxy, anda saturated, unsaturated, or aromatic heterocyclic group, may be furthersubstituted. In more particular embodiments, R¹ is a phenyl substitutedwith a halogen and a hydroxyl.

In some embodiments, the additional atoms or linkers necessary to attachthe metal chelating moiety to the rest of the compound comprises analkyl, aryl, combination of alkyl and aryl, or alkyl and aryl groupshaving heteroatoms. In certain embodiments, the additional atoms orlinkers necessary to attach the metal chelating moiety to the rest ofthe compound comprises an alkyl, wherein the alkyl may be substituted orunsubstituted.

As used herein, “alkyl” is intended to include branched, straight-chain,and cyclic saturated aliphatic hydrocarbon groups. Examples of alkylinclude, but are not limited to, methyl, ethyl, {circumflex over( )}-propyl, zso-propyl, «-butyl, sec-butyl, tert-butyl, «-pentyl, andsec-pentyl. In certain embodiments, alkyl groups are C1-C6 alkyl groupsor Ci-C4 alkyl groups. Particular alkyl groups are methyl, ethyl,propyl, butyl, and 3-pentyl. The term “Ci-C6 alkyl” as used herein meansstraight-chain, branched, or cyclic Ci-C6 hydrocarbons which arecompletely saturated and hybrids thereof such as (cycloalkyl)alkyl.Examples of Ci-C6 alkyl substituents include methyl (Me), ethyl (Et),propyl (including rø-propyl (n-Pr, nPr), wo-propyl (i-Pr, 1Pr), andcyclopropyl (c-Pr, 0Pr)), butyl (including rø-butyl (n-Bu, “Bu),zso-butyl (i-Bu, 1Bu), sec-butyl (s-Bu, sBu), tert-butyl (t-Bu, 1Bu), orcyclobutyl (c-Bu, 0Bu)), and so forth. “Cycloalkyl” is intended toinclude saturated ring groups, such as cyclopropyl, cyclobutyl,cyclopentyl, or cyclohexyl. Cycloalkyl groups typically will have 3 toabout 8 ring members. In the term “(cycloalkyl)alkyl”, cycloalkyl, andalkyl are as defined above, and the point of attachment is on the alkylgroup. This term encompasses, but is not limited to, cyclopropylmethyl,cyclopentylmethyl, and cyclohexylmethyl. The alkyl group may besubstituted or unsubstituted. Substituents are not counted towards thetotal number of atoms in the alkyl group, so long as the total atoms inthe substituent(s) are not larger than the alkyl group.

As used herein, the term “aryl” includes aromatic groups that contain 1to 3 separate or fused rings and from 2 to about 12 carbon atoms, and upto 3 heteroatoms as ring members. Examples of heteroatoms includenitrogen, oxygen or sulfur atoms. The aryl group may have 0, 1, 2 or 3heteroatoms as ring members. Examples of aryl groups include but are notlimited to phenyl, biphenyl and naphthyl, including 1-napthyl and2-naphthyl. Examples of aryl groups having heteroatoms includequinolinyl, isoquinolinyl, quinazolinyl, pyridyl, pyrazinyl, pyrimidyl,furanyl, pyrrolyl, thienyl, oxadiazolyl, thiadiazolyl, thiazolyl,triazinyl, oxazolyl, isoxazolyl, imidazolyl, indolyl, benzofiranyl, andbenzothiazolyl, among others. The aryl group may be substituted orunsubstituted. Substituents are not counted towards the total number ofatoms in the aryl group, so long as the total atoms in thesubstituent(s) are not larger than the aryl group.

As used herein, the term “alkylaryl” includes alkyl groups, as definedabove, substituted by aryl groups, as defined above. The aryl group maybe connected at any point on the alkyl group. The term C4-Cj6 alkylarylincludes alkylaryl groups having a total of 4 to 16 carbon atoms,counting the carbon atoms on the alkyl group and aryl group together.Examples of alkylaryl groups include but are not limited to benzyl(phenylmethyl), phenyl ethyl, and naphthylmethyl. The alkylaryl groupmay be substituted or unsubstituted. Substituents are not countedtowards the total number of atoms in the alkylaryl group, so long as thetotal atoms in the substituent(s) are not larger than the alkylarylgroup.

The term “substituted,” as used herein, means that any one or morehydrogens on the designated atom or group is replaced with asubstituent, provided that the designated atom's normal valence is notexceeded, and that the substitution results in a stable compound. When asubstituent is oxo (keto, i.e., =0), then 2 hydrogens on an atom arereplaced. The present invention is intended to include all isotopes(including radioisotopes) of atoms occurring in the present compounds.When the compounds are substituted, they may be so substituted at one ormore available positions, typically 1, 2, 3 or 4 positions, by one ormore suitable groups such as those disclosed herein. Suitable groupsthat may be present on a “substituted” group include e.g., halogen;cyano; hydroxyl; nitro; azido; amino; alkanoyl (such as a C1-C6 alkanoylgroup such as acyl or the like); carboxamido; alkyl groups (includingcycloalkyl groups, having 1 to about 8 carbon atoms, for example 1, 2,3, 4, 5, or 6 carbon atoms); alkenyl and alkynyl groups (includinggroups having one or more unsaturated linkages and from 2 to about 8,such as 2, 3, 4, 5 or 6, carbon atoms); alkoxy groups having one or moreoxygen linkages and from 1 to about 8, for example 1, 2, 3, 4, 5 or 6carbon atoms; aryloxy such as phenoxy; alkylthio groups including thosehaving one or more thioether linkages and from 1 to about 8 carbonatoms, for example 1, 2, 3, 4, 5 or 6 carbon atoms; alkylsulfinyl groupsincluding those having one or more sulfinyl linkages and from 1 to about8 carbon atoms, such as 1, 2, 3, 4, 5, or 6 carbon atoms; alkylsulfonylgroups including those having one or more sulfonyl linkages and from 1to about 8 carbon atoms, such as 1, 2, 3, 4, 5, or 6 carbon atoms;aminoalkyl groups including groups having one or more N atoms and from 1to about 8, for example 1, 2, 3, 4, 5 or 6, carbon atoms; carbocyclicaryl having 4, 5, 6 or more carbons and one or more rings, (e.g.,phenyl, biphenyl, naphthyl, or the like, each ring either substituted orunsubstituted aromatic); arylalkyl having 1 to 3 separate or fused ringsand from 6 to about 18 ring carbon atoms, (e.g. benzyl); arylalkoxyhaving 1 to 3 separate or fused rings and from 6 to about 18 ring carbonatoms (e.g. O-benzyl); or a saturated, unsaturated, or aromaticheterocyclic group having 1 to 3 separate or fused rings with 3 to about8 members per ring and one or more N, O or S atoms, (e.g. coumarinyl,quinolinyl, isoquinolinyl, quinazolinyl, pyridyl, pyrazinyl, pyrimidyl,furanyl, pyrrolyl, thienyl, thiazolyl, triazinyl, oxazolyl, isoxazolyl,imidazolyl, indolyl, benzofuranyl, benzothiazolyl, tetrahydrofuranyl,tetrahydropyranyl, piperidinyl, morpholinyl, piperazinyl, andpyrrolidinyl). Such heterocyclic groups may be further substituted, e.g.with hydroxy, alkyl, alkoxy, halogen and amino.

As used herein, where an internal substituent is flanked by bonds (forexample —NRC(O)—) the order of the atoms is fixed, the orientation ofthe group may not be reversed, and is inserted into a structure in theorientation presented. In other words —NRC(O)— is not the same as—C(O)NR—. As used herein the term C(O) (for example —NRC(O)—) is used toindicate a carbonyl (C═O) group, where the oxygen is bonded to thecarbon by a double bond.

In some embodiments, Ch comprises a structure selected from the groupconsisting of:

In particular embodiments, Ch comprises1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

In some embodiments, the compound of formula (I) is selected from thegroup consisting of:

In some embodiments, Ch includes a chelated metal and the chelated metalcomprises a radioactive isotope. In certain embodiments, the radioactiveisotope is Tc-99m, In-111, Ga-67, Ga-68, Y-86, Y-90, Lu-177, Re-186,Re-188, Cu-64, Cu-67, Co-55, Co-57, Sc-47, Ac-225, Bi-213, Bi-212,Pb-212, Sm-153, Ho-166, or Dy-166. In particular embodiments, theradioactive isotope is Ga-68 or Lu-177.

Other embodiments include pharmaceutically acceptable salts of thecompounds described in any of the previous embodiments. As used herein,“pharmaceutically acceptable salts” refer to derivatives of thedisclosed compounds wherein the parent compound is modified by makingnon-toxic acid or base salts thereof. Examples of pharmaceuticallyacceptable salts include, but are not limited to, mineral or organicacid salts of basic residues such as amines; alkali or organic salts ofacidic residues such as carboxylic acids; and the like. Thepharmaceutically acceptable salts include the conventional non-toxicsalts or the quaternary ammonium salts of the parent compound formed,for example, from non-toxic inorganic or organic acids. For example,conventional non-toxic acid salts include those derived from inorganicacids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric,nitric and the like; and the salts prepared from organic acids such asacetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric,citric, ascorbic. pamoic, malefic, hydroxymaleic, phenylacetic,glutamic, benzoic, salicylic. mesylic, sulfanilic. 2-acetoxybenzoic,fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic,isethionic, HOOC—(CH₂)_(n)—COOH where n is 0-4, and the like. Thepharmaceutically acceptable salts of the present invention can besynthesized from a parent compound that contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting free acid forms of these compounds with astoichiometric amount of the appropriate base (such as Na, Ca, Mg, or Khydroxide, carbonate, bicarbonate, or the like), or by reacting freebase forms of these compounds with a stoichiometric amount of theappropriate acid. Such reactions are typically carried out in water orin an organic solvent, or in a mixture of the two. Generally,non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, oracetonitrile are used, where practicable. Lists of additional suitablesalts may be found, e.g., in Remington's Pharmaceutical Sciences, 17thed., Mack Publishing Company, Easton, Pa., p. 1418 (1985).

The compounds described in the above embodiments may be made usingprocedures known in the art. In general, the materials used will bedetermined by the desired structure, and the type of linkage used.

Often, the compounds are prepared by sequentially adding components to apreformed urea, such as the lysine-urea-glutamate compounds described inBanerjee et al. (J. Med. Chem. vol. 51, pp. 4504-4517, 2008). Otherurea-based compounds may also be used as building blocks.

Compounds are assembled by reactions between different components, toform linkages such as ureas (—NRC(O)NR—), thioureas (—NRC(S)NR—), amides(—C(O)NR— or —NRC(O)—), or esters (—C(O)O— or —OC(O)—). Urea linkagesmay be readily prepared by reaction between an amine and an isocyanate,or between an amine and an activated carbonamide (—NRC(O)—). Thioureasmay be readily prepared from reaction of an amine with anisothiocyanate. Amides (—C(O)NR— or —NRC(O)—) may be readily prepared byreactions between amines and activated carboxylic acids or esters, suchas an acyl halide or N-hydroxysuccinimide ester. Carboxylic acids mayalso be activated in situ, for example, with a coupling reagent, such asa carbodiimide, or carbonyldiimidazole (CDI). Esters may be formed byreaction between alcohols and activated carboxylic acids. Triazoles arereadily prepared by reaction between an azide and an alkyne. optionallyin the presence of a copper (Cu) catalyst.

Protecting groups may be used, if necessary. to protect reactive groupswhile the compounds are being assembled. Suitable protecting groups, andtheir removal, will be readily available to one of ordinary skill in theart.

In this way, the compounds may be easily prepared from individualbuilding blocks, such as amines, carboxylic acids, and amino acids.

Often, a Ch or FB group is placed on the compound by adding a metalchelating group or fluorescent dye to the compound toward the end of asynthesis, for example by reacting a reactive amine on the compound withan activated metal chelating group or fluorescent dye. A wide variety ofmetal chelating groups and fluorescent dyes are known in the art, withactivated functional groups for reacting with amines. The type of metalchelating group will be determined, in part by the desired metal.Selecting a metal chelating group for a particular metal atom will beapparent to one of ordinary skill in the art. The fluorescent dye usedwith be determined, in part, by the desired wavelength of fluorescence,and may be readily selected by one of ordinary skill in the art.

Exemplary procedures for specific compounds are described in theExamples below. Other compounds within the scope of the claims can beprepared using readily apparent modifications of these procedures.

G.2 Uses

Compounds described above, including various radiolabeled compounds, maybe used for diagnostic, imaging, or therapeutic purposes. In general,the suitability of a particular radioisotope for a particular purpose(i.e. imaging or therapeutic) is well understood in the art. Otherexemplary embodiments are compounds used as precursors for radiolabeledcompounds, in which a metal or radioactive isotope of a metal may beadded to the compound. Some compounds according to the invention areintermediates for forming other compounds of the invention.

G.2.1 Imaging

Embodiments include methods of imaging one or more cells, organs ortissues comprising exposing cells to or administering to a subject aneffective amount of a compound with an isotopic label suitable forimaging. In some embodiments, the one or more organs or tissues includeprostate tissue, kidney tissue, brain tissue, vascular tissue or tumortissue. The cells, organs or tissues may be imaged while within anorganism, either by whole body imaging or intraoperative imaging, or maybe excised from the organism for imaging.

In another embodiment, the imaging method is suitable for imagingstudies of PSMA inhibitors, for example, by studying competitive bindingof non-radiolabeled inhibitors. In still another embodiment, the imagingmethod is suitable for imaging of cancer, tumor or neoplasm. In afurther embodiment, the cancer is selected from eye or ocular cancer,rectal cancer, colon cancer, cervical cancer, prostate cancer, breastcancer and bladder cancer, oral cancer, benign and malignant tumors,stomach cancer, liver cancer, pancreatic cancer, lung cancer, corpusuteri, ovary cancer, prostate cancer, testicular cancer, renal cancer,brain cancer (e.g., gliomas), throat cancer, skin melanoma, acutelymphocytic leukemia, acute myelogenous leukemia. Ewing's Sarcoma,Kaposi's Sarcoma, basal cell carcinoma and squamous cell carcinoma,small cell lung cancer, choriocarcinoma, rhabdomyosarcoma, angiosarcoma,hemangioendothelioma, Wilms Tumor, neuroblastoma, mouth/pharynx cancer,esophageal cancer, larynx cancer, lymphoma, neurofibromatosis, tuberoussclerosis, hemangiomas, and lymphangiogenesis.

The imaging methods of the invention are suitable for imaging anyphysiological process or feature in which PSMA is involved. Typically,imaging methods are suitable for identification of areas of tissues ortargets which express high concentrations of PSMA. Typical applicationsinclude imaging glutamateric neurotransmission, presynapticglutamatergic neurotransmission, malignant tumors or cancers thatexpress PSMA, prostate cancer (including metastasized prostate cancer),and angiogenesis. Essentially all solid tumors express PSMA in theneovasculture. Therefore, methods of the present invention can be usedto image nearly all solid tumors including lung, renal cell,glioblastoma, pancreas, bladder, sarcoma, melanoma, breast, colon, germcell, pheochromocytoma, esophageal and stomach. Also, certain benignlesions and tissues including endometrium, schwannoma and Barrett'sesophagus can be imaged according to the present invention.

The methods of imaging angiogenesis are suitable for use in imaging avariety of diseases and disorders in which angiogenesis takes place.Illustrative, non-limiting, examples include tumors, collagen vasculardisease, cancer, stroke, vascular malformations, and retinopathy.Methods of imaging angiogenesis are also suitable for use in diagnosisand observation of normal tissue development.

PSMA is frequently expressed in endothelial cells of capillary vesselsin peritumoral and endotumoral areas of various malignancies such thatcompounds of the invention and methods of imaging using same aresuitable for imaging such malignancies. In certain embodiments, theradiolabeled compound is stable in vivo.

In certain embodiments, the radiolabeled compound is detectable bypositron emission tomography (PET) or single photon emission computedtomography (SPECT). [00170] In some embodiments, the subject is a human,rat, mouse, cat, dog, horse, sheep, cow, monkey, avian, or amphibian. Inanother embodiment, the cell is in vivo or in vitro. Typical subjects towhich compounds of the invention may be administered will be mammals,particularly primates, especially humans. For veterinary applications, awide variety of subjects will be suitable, e. g. livestock such ascattle, sheep, goats, cows, swine and the like; poultry such aschickens, ducks, geese, turkeys, and the like; and domesticated animalsparticularly pets such as dogs and cats. For diagnostic or researchapplications, a wide variety of mammals will be suitable subjectsincluding rodents (e.g. mice, rats, hamsters), rabbits, primates, andswine such as inbred pigs and the like. Additionally, for in vitroapplications, such as in vitro diagnostic and research applications,body fluids and cell samples of the above subjects will be suitable foruse such as mammalian, particularly primate such as human, blood, urineor tissue samples, or blood urine or tissue samples of the animalsmentioned for veterinary applications. In other in vitro applications,the cells or tissues are present in culture or in suspension.

Imaging agents of the invention may be used in accordance with themethods of the invention by one of skill in the art. Images can begenerated by virtue of differences in the spatial distribution of theimaging agents which accumulate at a site when contacted with PSMA. Thespatial distribution may be measured using any means suitable for theparticular label, for example, a gamma camera, a PET apparatus, a SPECTapparatus, a fluorescence camera and the like. The extent ofaccumulation of the imaging agent may be quantified using known methodsfor quantifying radioactive emissions. A particularly useful imagingapproach employs more than one imaging agent, or a bimodal agent havinga fluorescent dye moiety and a metal chelating group, such as thosedescribed above, to perform simultaneous studies.

In general, a detectably effective amount of the imaging agent isadministered to a subject. As used herein, “a detectably effectiveamount” of the imaging agent is defined as an amount sufficient to yieldan acceptable image using equipment which is available for clinical use.A detectably effective amount of the imaging agent may be administeredin more than one injection. The detectably effective amount of theimaging agent can vary according to factors such as the degree ofsusceptibility of the individual, the age, sex, and weight of theindividual, idiosyncratic responses of the individual, and thedosimetry. Detectably effective amounts of the imaging agent can alsovary according to instrument and film-related factors. Optimization ofsuch factors is well within the level of skill in the art. The amount ofimaging agent used for diagnostic purposes and the duration of theimaging study will depend upon the radionuclide used to label the agent,the body mass of the patient, the nature and severity of the conditionbeing treated, the nature of therapeutic treatments which the patienthas undergone, and on the idiosyncratic responses of the patient.Ultimately, the attending physician will decide the amount of imagingagent to administer to each individual patient and the duration of theimaging study.

In some embodiments, the compounds are excreted from tissues of the bodyquickly to prevent prolonged exposure to the radiation of theradiolabeled compound administered to the patient. Generally, thecompounds are excreted from tissues of the body slowly enough to allowsufficient time for imaging or other use. Typically compounds of theinvention are eliminated from the body in less than about 24 hours. Moretypically, compounds of the invention are eliminated from the body inless than about 16 hours, 12 hours, 8 hours, 6 hours, 4 hours, 2 hours,90 minutes, or 60 minutes. Exemplary compounds are eliminated in betweenabout 60 minutes and about 120 minutes.

In some embodiments of the invention, the compounds are designed toincrease uptake in PSMA positive cells (i.e. tumor cells). For example,highly hydrophilic compounds may be excreted quickly. Compounds withincreased hydrophobicity. such as compounds having hydrophobic linkers,may have longer circulation times, thereby providing more prolongedsupply of tracer to bind to cells. According to embodiments of compoundsaccording to the invention, hydrophobicity can be increased when, forexample, p is 1 or more, or when R² or R³ is CO₂R⁴

Accordingly, in some embodiments, the presently disclosed subject matterprovides a method for imaging one or more prostate-specific membraneantigen (PSMA) tumors, or cells the method comprising contacting the oneor more tumors, or cells, with an effective amount of a compound offormula (I), or a pharmaceutically acceptable salt thereof, and makingan image.

In some embodiments, the imaging comprises positron emission tomography(PET).

In some embodiments, the one or more PSMA-expressing tumors or cells isselected from the group consisting of a prostate tumor or cell, ametastasized prostate tumor or cell, a lung tumor or cell, a renal tumoror cell, a glioblastoma, a pancreatic tumor or cell, a bladder tumor orcell, a sarcoma, a melanoma, a breast tumor or cell, a colon tumor orcell, a germ cell, a pheochromocytoma, an esophageal tumor or cell, astomach tumor or cell, and combinations thereof.

G.2.2 Therapeutic Uses

Embodiments of the invention include methods of treating a tumorcomprising administering a therapeutically effective amount of acompound discussed above, where the compound includes a therapeuticallyeffective radioisotope. The development of low molecular weightradiotherapeutic agents is much different from developingradiopharmaceuticals for imaging in that longer tumor residence timesmay be important for the former.

In some embodiments, the tumor cells may express PSMA, such as prostatetumor cells or metastasized prostate tumor cells. In other embodiments,a tumor may be treated by targeting adjacent or nearby cells whichexpress PSMA. For example, vascular cells undergoing angiogenesisassociated with a tumor may be targeted. Essentially all solid tumorsexpress PSMA in the neovasculture. Therefore, methods of the presentinvention can be used to treat nearly all solid tumors including lung,renal cell, glioblastoma, pancreas, bladder, sarcoma, melanoma, breast,colon, germ cell, pheochromocytoma. esophageal and stomach. Also,certain benign lesions and tissues including endometrium, schwannoma andBarrett's esophagus can be treated according to the present invention.Examples of therapeutically effective radioisotopes include ⁹⁰Y, ¹⁷⁷Lu,¹⁸⁶Re, ¹⁸⁸Re, ⁶⁷Cu, ²²⁵Ac, ²¹³Bi, ²¹²Br ⁶⁷Ga, ¹¹¹In, ¹⁵³Sm, ²¹²Pb ¹³¹Iand ²¹¹At.

Accordingly, in some embodiments, the presently disclosed subject matterprovides a method for treating a tumor comprising administering atherapeutically effective amount of a compound of formula (I), or apharmaceutically acceptable salt thereof, wherein the compound includesa therapeutically effective radioisotope.

G.3 Pharmaceutical Compositions and Kits

The compounds discussed herein can be formulated into variouscompositions, for use in diagnostic, imaging or therapeutic treatmentmethods. The compositions (e.g. pharmaceutical compositions) can beassembled as a kit. Generally, a pharmaceutical composition comprises aneffective amount (e.g., a pharmaceutically effective amount, ordetectably effective amount) of a compound described above.

A composition of the invention can be formulated as a pharmaceuticalcomposition, which comprises a compound of the invention andpharmaceutically acceptable carrier. By a “pharmaceutically acceptablecarrier” is meant a material that is not biologically or otherwiseundesirable, i.e., the material may be administered to a subject withoutcausing any undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained. The carrier wouldnaturally be selected to minimize any degradation of the activeingredient and to minimize any adverse side effects in the subject, aswould be well known to one of skill in the art. For a discussion ofpharmaceutically acceptable carriers and other components ofpharmaceutical compositions, see, e.g., Remington's PharmaceuticalSciences, 18¹ ed., Mack Publishing Company, 1990. Some suitablepharmaceutical carriers will be evident to a skilled worker and include,e.g., water (including sterile and/or deionized water), suitable buffers(such as PBS), physiological saline, cell culture medium (such as DMEM),artificial cerebral spinal fluid, or the like.

A pharmaceutical composition or kit of the invention can contain otherpharmaceuticals, in addition to the compound. The other agent(s) can beadministered at any suitable time during the treatment of the patient,either concurrently or sequentially. [00183] One skilled in the art willappreciate that the particular formulation will depend, in part, uponthe particular agent that is employed, and the chosen route ofadministration. Accordingly, there is a wide variety of suitableformulations of compositions of the present invention.

One skilled in the art will appreciate that a suitable or appropriateformulation can be selected, adapted or developed based upon theparticular application at hand. Dosages for compositions of theinvention can be in unit dosage form. The term “unit dosage form” asused herein refers to physically discrete units suitable as unitarydosages for animal (e.g. human) subjects, each unit containing apredetermined quantity of an agent of the invention, alone or incombination with other therapeutic agents, calculated in an amountsufficient to produce the desired effect in association with apharmaceutically acceptable diluent, carrier, or vehicle. [00185] Oneskilled in the art can easily determine the appropriate dose, schedule,and method of administration for the exact formulation of thecomposition being used, in order to achieve the desired effective amountor effective concentration of the agent in the individual patient.

The dose of a composition of the invention, administered to an animal.particularly a human, in the context of the present invention should besufficient to produce at least a detectable amount of a diagnostic ortherapeutic response in the individual over a reasonable time frame. Thedose used to achieve a desired effect will be determined by a variety offactors, including the potency of the particular agent beingadministered, the pharmacodynamics associated with the agent in thehost, the severity of the disease state of infected individuals, othermedications being administered to the subject, etc. The size of the dosealso will be determined by the existence of any adverse side effectsthat may accompany the particular agent, or composition thereof,employed. It is generally desirable, whenever possible, to keep adverseside effects to a minimum. The dose of the biologically active materialwill vary; suitable amounts for each particular agent will be evident toa skilled worker.

Other embodiments provide kits including a compound according to theinvention. In certain embodiments, the kit provides packagedpharmaceutical compositions having a pharmaceutically acceptable carrierand a compound of the invention. In some embodiments the packagedpharmaceutical composition will include the reaction precursorsnecessary to generate the compound of the invention upon combinationwith a radionuclide. Other packaged pharmaceutical compositions providedby the present invention further comprise indicia comprising at leastone of; instructions for preparing compounds according to the inventionfrom supplied precursors, instructions for using the composition toimage cells or tissues expressing PSMA, or instructions for using thecomposition to image glutamatergic neurotransmission in a patientsuffering from a stress-related disorder, or instructions for using thecomposition to image prostate cancer.

In certain embodiments, a kit according to the invention contains fromabout 1 mCi to about 30 mCi of the radionuclide-labeled imaging agentdescribed above, in combination with a pharmaceutically acceptablecarrier. The imaging agent and carrier may be provided in solution or inlyophilized form. When the imaging agent and carrier of the kit are inlyophilized form, the kit may optionally contain a sterile andphysiologically acceptable reconstitution medium such as water, saline,buffered saline, and the like. The kit may provide a compound of theinvention in solution or in lyophilized form, and these components ofthe kit of the invention may optionally contain stabilizers such asNaCl, silicate, phosphate buffers, ascorbic acid, gentisic acid, and thelike. Additional stabilization of kit components may be provided in thisembodiment, for example, by providing the reducing agent in anoxidation-resistant form.

Determination and optimization of such stabilizers and stabilizationmethods are well within the level of skill in the art.

A “pharmaceutically acceptable carrier” refers to a biocompatiblesolution, having due regard to sterility, p[Eta], isotonicity,stability, and the like and can include any and all solvents, diluents(including sterile saline, Sodium Chloride Injection, Ringer'sInjection, Dextrose Injection, Dextrose and Sodium Chloride Injection.Lactated Ringer's Injection and other aqueous buffer solutions),dispersion media, coatings, antibacterial and antifungal agents,isotonic agents, and the like. The pharmaceutically acceptable carriermay also contain stabilizers, preservatives, antioxidants, or otheradditives, which are well known to one of skill in the art, or othervehicle as known in the art.

Accordingly, in some embodiments, the presently disclosed subject matterprovides a kit comprising a compound of formula (I), or apharmaceutically acceptable salt thereof.

XXX II. Definitions

Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this presently described subject matter belongs.

While the following terms in relation to the presently disclosedcompounds are believed to be well understood by one of ordinary skill inthe art, the following definitions are set forth to facilitateexplanation of the presently disclosed subject matter. These definitionsare intended to supplement and illustrate, not preclude, the definitionsthat would be apparent to one of ordinary skill in the art upon reviewof the present disclosure.

The terms substituted, whether preceded by the term “optionally” or not,and substituent, as used herein, refer to the ability, as appreciated byone skilled in this art, to change one functional group for anotherfunctional group on a molecule, provided that the valency of all atomsis maintained. When more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. The substituents also may be further substituted (e.g., anaryl group substituent may have another substituent off it, such asanother aryl group, which is further substituted at one or morepositions).

Where substituent groups or linking groups are specified by theirconventional chemical formulae, written from left to right, they equallyencompass the chemically identical substituents that would result fromwriting the structure from right to left, e.g., —CH₂O— is equivalent to—OCH₂—; —C(═O)O— is equivalent to —OC(═O)—; —OC(═O)NR— is equivalent to—NRC(═O)O—, and the like.

When the term “independently selected” is used, the substituents beingreferred to (e.g., R groups, such as groups R₁, R₂, and the like, orvariables, such as “m” and “n”), can be identical or different. Forexample, both R₁ and R₂ can be substituted alkyls, or R₁ can be hydrogenand R₂ can be a substituted alkyl, and the like.

The terms “a,” “an,” or “a(n),” when used in reference to a group ofsubstituents herein, mean at least one. For example, where a compound issubstituted with “an” alkyl or aryl, the compound is optionallysubstituted with at least one alkyl and/or at least one aryl. Moreover,where a moiety is substituted with an R substituent, the group may bereferred to as “R-substituted.” Where a moiety is R-substituted, themoiety is substituted with at least one R substituent and each Rsubstituent is optionally different.

A named “R” or group will generally have the structure that isrecognized in the art as corresponding to a group having that name,unless specified otherwise herein. For the purposes of illustration,certain representative “R” groups as set forth above are defined below.

Description of compounds of the present disclosure is limited byprinciples of chemical bonding known to those skilled in the art.Accordingly, where a group may be substituted by one or more of a numberof substituents, such substitutions are selected so as to comply withprinciples of chemical bonding and to give compounds which are notinherently unstable and/or would be known to one of ordinary skill inthe art as likely to be unstable under ambient conditions, such asaqueous, neutral, and several known physiological conditions. Forexample, a heterocycloalkyl or heteroaryl is attached to the remainderof the molecule via a ring heteroatom in compliance with principles ofchemical bonding known to those skilled in the art thereby avoidinginherently unstable compounds.

Unless otherwise explicitly defined, a “substituent group,” as usedherein, includes a functional group selected from one or more of thefollowing moieties, which are defined herein:

The term hydrocarbon, as used herein, refers to any chemical groupcomprising hydrogen and carbon. The hydrocarbon may be substituted orunsubstituted. As would be known to one skilled in this art, allvalencies must be satisfied in making any substitutions. The hydrocarbonmay be unsaturated, saturated, branched, unbranched, cyclic, polycyclic,or heterocyclic. Illustrative hydrocarbons are further defined hereinbelow and include, for example, methyl, ethyl, n-propyl, isopropyl,cyclopropyl, allyl, vinyl, n-butyl, tert-butyl, ethynyl, cyclohexyl, andthe like.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e., unbranched) or branchedchain, acyclic or cyclic hydrocarbon group, or combination thereof,which may be fully saturated, mono- or polyunsaturated and can includedi- and multivalent groups, having the number of carbon atoms designated(i.e., C₁-C₁₀ means one to ten carbons, including 1, 2, 3, 4, 5, 6, 7,8, 9, and 10 carbons). In particular embodiments, the term “alkyl”refers to C₁₋₂₀ inclusive, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, and 20 carbons, linear (i.e.,“straight-chain”), branched, or cyclic, saturated or at least partiallyand in some embodiments fully unsaturated (i.e., alkenyl and alkynyl)hydrocarbon radicals derived from a hydrocarbon moiety containingbetween one and twenty carbon atoms by removal of a single hydrogenatom.

Representative saturated hydrocarbon groups include, but are not limitedto, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,tert-butyl, n-pentyl, sec-pentyl, isopentyl, neopentyl, n-hexyl,sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, and homologs and isomers thereof.

“Branched” refers to an alkyl group in which a lower alkyl group, suchas methyl, ethyl or propyl, is attached to a linear alkyl chain. “Loweralkyl” refers to an alkyl group having 1 to about 8 carbon atoms (i.e.,a C₁₋₈ alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. “Higheralkyl” refers to an alkyl group having about 10 to about 20 carbonatoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms.In certain embodiments, “alkyl” refers, in particular, to C₁₋₈straight-chain alkyls. In other embodiments, “alkyl” refers, inparticular, to C₁₋₈ branched-chain alkyls.

Alkyl groups can optionally be substituted (a “substituted alkyl”) withone or more alkyl group substituents, which can be the same ordifferent. The term “alkyl group substituent” includes but is notlimited to alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl,aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio,carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. There can be optionallyinserted along the alkyl chain one or more oxygen, sulfur or substitutedor unsubstituted nitrogen atoms, wherein the nitrogen substituent ishydrogen, lower alkyl (also referred to herein as “alkylaminoalkyl”), oraryl.

Thus, as used herein, the term “substituted alkyl” includes alkylgroups, as defined herein, in which one or more atoms or functionalgroups of the alkyl group are replaced with another atom or functionalgroup, including for example, alkyl, substituted alkyl, halogen, aryl,substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino,dialkylamino, sulfate, and mercapto.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon group, or combinations thereof, consisting of atleast one carbon atoms and at least one heteroatom selected from thegroup consisting of O, N, P, Si and S, and wherein the nitrogen,phosphorus, and sulfur atoms may optionally be oxidized and the nitrogenheteroatom may optionally be quaternized. The heteroatom(s) O, N, P andS and Si may be placed at any interior position of the heteroalkyl groupor at the position at which alkyl group is attached to the remainder ofthe molecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃,—CH₂—CH₂₅—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃,—CH₂—CH═N—OCH₃, —CH═CH—N(CH₃)— CH₃, O—CH₃, —O—CH₂—CH₃, and —CN. Up totwo or three heteroatoms may be consecutive, such as, for example,—CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃.

As described above, heteroalkyl groups, as used herein, include thosegroups that are attached to the remainder of the molecule through aheteroatom, such as —C(O)NR′, —NR′R″, —OR′, —SR, —S(O)R, and/or—S(O₂)R′. Where “heteroalkyl” is recited, followed by recitations ofspecific heteroalkyl groups, such as —NR′R or the like, it will beunderstood that the terms heteroalkyl and —NR′R″ are not redundant ormutually exclusive. Rather, the specific heteroalkyl groups are recitedto add clarity.

Thus, the term “heteroalkyl” should not be interpreted herein asexcluding specific heteroalkyl groups, such as —NR′R″ or the like.

“Cyclic” and “cycloalkyl” refer to a non-aromatic mono- or multicyclicring system of about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8,9, or 10 carbon atoms. The cycloalkyl group can be optionally partiallyunsaturated. The cycloalkyl group also can be optionally substitutedwith an alkyl group substituent as defined herein, oxo, and/or alkylene.There can be optionally inserted along the cyclic alkyl chain one ormore oxygen, sulfur or substituted or unsubstituted nitrogen atoms,wherein the nitrogen substituent is hydrogen, unsubstituted alkyl,substituted alkyl, aryl, or substituted aryl, thus providing aheterocyclic group. Representative monocyclic cycloalkyl rings includecyclopentyl, cyclohexyl, and cycloheptyl. Multicyclic cycloalkyl ringsinclude adamantyl, octahydronaphthyl, decalin, camphor, camphane, andnoradamantyl, and fused ring systems, such as dihydro- andtetrahydronaphthalene, and the like.

The term “cycloalkylalkyl,” as used herein, refers to a cycloalkyl groupas defined hereinabove, which is attached to the parent molecular moietythrough an alkyl group, also as defined above. Examples ofcycloalkylalkyl groups include cyclopropylmethyl and cyclopentylethyl.

The terms “cycloheteroalkyl” or “heterocycloalkyl” refer to anon-aromatic ring system, unsaturated or partially unsaturated ringsystem, such as a 3- to 10-member substituted or unsubstitutedcycloalkyl ring system, including one or more heteroatoms, which can bethe same or different, and are selected from the group consisting ofnitrogen (N), oxygen (O), sulfur (S), phosphorus (P), and silicon (Si),and optionally can include one or more double bonds.

The cycloheteroalkyl ring can be optionally fused to or otherwiseattached to other cycloheteroalkyl rings and/or non-aromatic hydrocarbonrings. Heterocyclic rings include those having from one to threeheteroatoms independently selected from oxygen, sulfur, and nitrogen, inwhich the nitrogen and sulfur heteroatoms may optionally be oxidized andthe nitrogen heteroatom may optionally be quaternized. In certainembodiments, the term heterocylic refers to a non-aromatic 5-, 6-, or7-membered ring or a polycyclic group wherein at least one ring atom isa heteroatom selected from O, S, and N (wherein the nitrogen and sulfurheteroatoms may be optionally oxidized), including, but not limited to,a bi- or tri-cyclic group, comprising fused six-membered rings havingbetween one and three heteroatoms independently selected from theoxygen, sulfur, and nitrogen, wherein (i) each 5-membered ring has 0 to2 double bonds, each 6-membered ring has 0 to 2 double bonds, and each7-membered ring has 0 to 3 double bonds, (ii) the nitrogen and sulfurheteroatoms may be optionally oxidized, (iii) the nitrogen heteroatommay optionally be quaternized, and (iv) any of the above heterocyclicrings may be fused to an aryl or heteroaryl ring. Representativecycloheteroalkyl ring systems include, but are not limited topyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl,pyrazolinyl, piperidyl, piperazinyl, indolinyl, quinuclidinyl,morpholinyl, thiomorpholinyl, thiadiazinanyl, tetrahydrofuranyl, and thelike.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. The terms “cycloalkylene”and “heterocycloalkylene” refer to the divalent derivatives ofcycloalkyl and heterocycloalkyl, respectively.

An unsaturated alkyl group is one having one or more double bonds ortriple bonds. Examples of unsaturated alkyl groups include, but are notlimited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. Alkyl groups that arelimited to hydrocarbon groups are termed “homoalkyl.”

More particularly, the term “alkenyl” as used herein refers to amonovalent group derived from a C₁₋₂₀ inclusive straight or branchedhydrocarbon moiety having at least one carbon-carbon double bond by theremoval of a single hydrogen molecule. Alkenyl groups include, forexample, ethenyl (i.e., vinyl), propenyl, butenyl,1-methyl-2-buten-1-yl, pentenyl, hexenyl, octenyl, allenyl, andbutadienyl.

The term “cycloalkenyl” as used herein refers to a cyclic hydrocarboncontaining at least one carbon-carbon double bond. Examples ofcycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclopentadiene, cyclohexenyl, 1,3-cyclohexadiene, cycloheptenyl,cycloheptatrienyl, and cyclooctenyl.

The term “alkynyl” as used herein refers to a monovalent group derivedfrom a straight or branched C₁₋₂₀ hydrocarbon of a designed number ofcarbon atoms containing at least one carbon-carbon triple bond. Examplesof “alkynyl” include ethynyl, 2-propynyl (propargyl), 1-propynyl,pentynyl, hexynyl, and heptynyl groups, and the like.

The term “alkylene” by itself or a part of another substituent refers toa straight or branched bivalent aliphatic hydrocarbon group derived froman alkyl group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbonatoms. The alkylene group can be straight, branched or cyclic. Thealkylene group also can be optionally unsaturated and/or substitutedwith one or more “alkyl group substituents.” There can be optionallyinserted along the alkylene group one or more oxygen, sulfur orsubstituted or unsubstituted nitrogen atoms (also referred to herein as“alkylaminoalkyl”), wherein the nitrogen substituent is alkyl aspreviously described. Exemplary alkylene groups include methylene(—CH₂—); ethylene (—CH₂—CH₂—); propylene (—(CH₂)₃—); cyclohexylene(—C₆H₁₀—); —CH═CH—CH═CH—; —CH═CH—CH₂—; —CH₂CH₂CH₂CH₂—, —CH₂CH═CHCH₂—,—CH₂CsCCH₂—, —CH₂CH₂CH(CH₂CH₂CH₃)CH₂—, —(CH₂)_(q)—N(R)—(CH₂)_(r)—,wherein each of q and r is independently an integer from 0 to about 20,e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20, and R is hydrogen or lower alkyl; methylenedioxyl(—O—CH₂—O—); and ethylenedioxyl (—O—(CH₂)₂—O—). An alkylene group canhave about 2 to about 3 carbon atoms and can further have 6-20 carbons.

Typically, an alkyl (or alkylene) group will have from 1 to 24 carbonatoms, with those groups having 10 or fewer carbon atoms being someembodiments of the present disclosure. A “lower alkyl” or “loweralkylene” is a shorter chain alkyl or alkylene group, generally havingeight or fewer carbon atoms.

The term “heteroalkylene” by itself or as part of another substituentmeans a divalent group derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms also can occupy either or both of thechain termini (e.g., alkyleneoxo, alkylenedioxo, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)OR′— represents both —C(O)OR′-and —R′OC(O)—.

The term “aryl” means, unless otherwise stated, an aromatic hydrocarbonsubstituent that can be a single ring or multiple rings (such as from 1to 3 rings), which are fused together or linked covalently. The term“heteroaryl” refers to aryl groups (or rings) that contain from one tofour heteroatoms (in each separate ring in the case of multiple rings)selected from N, O, and S, wherein the nitrogen and sulfur atoms areoptionally oxidized, and the nitrogen atom(s) are optionallyquaternized. A heteroaryl group can be attached to the remainder of themolecule through a carbon or heteroatom. Non-limiting examples of aryland heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl,4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of above noted aryland heteroaryl ring systems are selected from the group of acceptablesubstituents described below. The terms “arylene” and “heteroarylene”refer to the divalent forms of aryl and heteroaryl, respectively.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the terms “arylalkyl” and“heteroarylalkyl” are meant to include those groups in which an aryl orheteroaryl group is attached to an alkyl group (e.g., benzyl, phenethyl,pyridylmethyl, furylmethyl, and the like) including those alkyl groupsin which a carbon atom (e.g., a methylene group) has been replaced by,for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl,3-(1-naphthyloxy)propyl, and the like). However, the term “haloaryl,” asused herein is meant to cover only aryls substituted with one or morehalogens.

Where a heteroalkyl, heterocycloalkyl, or heteroaryl includes a specificnumber of members (e.g. “3 to 7 membered”), the term “member” refers toa carbon or heteroatom.

Further, a structure represented generally by the formula:

as used herein refers to a ring structure, for example, but not limitedto a 3-carbon, a 4-carbon, a 5-carbon, a 6-carbon, a 7-carbon, and thelike, aliphatic and/or aromatic cyclic compound, including a saturatedring structure, a partially saturated ring structure, and an unsaturatedring structure, comprising a substituent R group, wherein the R groupcan be present or absent, and when present, one or more R groups caneach be substituted on one or more available carbon atoms of the ringstructure. The presence or absence of the R group and number of R groupsis determined by the value of the variable “n,” which is an integergenerally having a value ranging from 0 to the number of carbon atoms onthe ring available for substitution. Each R group, if more than one, issubstituted on an available carbon of the ring structure rather than onanother R group. For example, the structure above where n is 0 to 2would comprise compound groups including, but not limited to:

and the like.

A dashed line representing a bond in a cyclic ring structure indicatesthat the bond can be either present or absent in the ring. That is, adashed line representing a bond in a cyclic ring structure indicatesthat the ring structure is selected from the group consisting of asaturated ring structure, a partially saturated ring structure, and anunsaturated ring structure.

The symbol (

) denotes the point of attachment of a moiety to the remainder of themolecule.

When a named atom of an aromatic ring or a heterocyclic aromatic ring isdefined as being “absent,” the named atom is replaced by a direct bond.

Each of above terms (e.g., “alkyl,” “heteroalkyl,” “cycloalkyl, and“heterocycloalkyl”, “aryl,” “heteroaryl,” “phosphonate,” and “sulfonate”as well as their divalent derivatives) are meant to include bothsubstituted and unsubstituted forms of the indicated group. Optionalsubstituents for each type of group are provided below.

Substituents for alkyl, heteroalkyl, cycloalkyl, heterocycloalkylmonovalent and divalent derivative groups (including those groups oftenreferred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl,alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —C(O)NR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)OR′,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such groups. R′, R″, R′″ and R″″ each mayindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g.,aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl,alkoxy or thioalkoxy groups, or arylalkyl groups. As used herein, an“alkoxy” group is an alkyl attached to the remainder of the moleculethrough a divalent oxygen. When a compound of the disclosure includesmore than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R′″ and R″″ groups when morethan one of these groups is present. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 4-, 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant toinclude, but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. Fromthe above discussion of substituents, one of skill in the art willunderstand that the term “alkyl” is meant to include groups includingcarbon atoms bound to groups other than hydrogen groups, such ashaloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃,—C(O)CH₂OCH₃, and the like).

Similar to the substituents described for alkyl groups above, exemplarysubstituents for aryl and heteroaryl groups (as well as their divalentderivatives) are varied and are selected from, for example: halogen,—OR′, —NR′R″, —SR′, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —C(O)NR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)OR′,—NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″,—NRSO₂R′, —CN and —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxo, andfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total number ofopen valences on aromatic ring system; and where R′, R″, R′″ and R″″ maybe independently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl and substituted or unsubstitutedheteroaryl. When a compound of the disclosure includes more than one Rgroup, for example, each of the R groups is independently selected asare each R′, R″, R′″ and R″″ groups when more than one of these groupsis present.

Two of the substituents on adjacent atoms of aryl or heteroaryl ring mayoptionally form a ring of the formula -T-C(O)—(CRR′)_(q)—U—, wherein Tand U are independently —NR—, —O—, —CRR′— or a single bond, and q is aninteger of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂)_(r)—B—, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or asingle bond, and r is an integer of from 1 to 4.

One of the single bonds of the new ring so formed may optionally bereplaced with a double bond. Alternatively, two of the substituents onadjacent atoms of aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula —(CRR′)_(s)—X′— (C″R′″)_(d)—, where sand d are independently integers of from 0 to 3, and X′ is —O—, —NR′—,—S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—. The substituents R, R′, R″ and R′″may be independently selected from hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl.

As used herein, the term “acyl” refers to an organic acid group whereinthe —OH of the carboxyl group has been replaced with another substituentand has the general formula RC(═O)—, wherein R is an alkyl, alkenyl,alkynyl, aryl, carbocylic, heterocyclic, or aromatic heterocyclic groupas defined herein). As such, the term “acyl” specifically includesarylacyl groups, such as a 2-(furan-2-yl)acetyl)- and a 2-phenylacetylgroup. Specific examples of acyl groups include acetyl and benzoyl. Acylgroups also are intended to include amides, —RC(═O)NR′, esters,—RC(═O)OR′, ketones, —RC(═O)R′, and aldehydes, —RC(═O)H.

The terms “alkoxyl” or “alkoxy” are used interchangeably herein andrefer to a saturated (i.e., alkyl-O—) or unsaturated (i.e., alkenyl-O—and alkynyl-O—) group attached to the parent molecular moiety through anoxygen atom, wherein the terms “alkyl,” “alkenyl,” and “alkynyl” are aspreviously described and can include C₁₋₂₀ inclusive, linear, branched,or cyclic, saturated or unsaturated oxo-hydrocarbon chains, including,for example, methoxyl, ethoxyl, propoxyl, isopropoxyl, n-butoxyl,sec-butoxyl, tert-butoxyl, and n-pentoxyl, neopentoxyl, n-hexoxyl, andthe like.

The term “alkoxyalkyl” as used herein refers to an alkyl-O-alkyl ether,for example, a methoxyethyl or an ethoxymethyl group.

“Aryloxyl” refers to an aryl-O— group wherein the aryl group is aspreviously described, including a substituted aryl. The term “aryloxyl”as used herein can refer to phenyloxyl or hexyloxyl, and alkyl,substituted alkyl, halo, or alkoxyl substituted phenyloxyl or hexyloxyl.

“Aralkyl” refers to an aryl-alkyl-group wherein aryl and alkyl are aspreviously described, and included substituted aryl and substitutedalkyl. Exemplary aralkyl groups include benzyl, phenylethyl, andnaphthylmethyl.

“Aralkyloxyl” refers to an aralkyl-O— group wherein the aralkyl group isas previously described. An exemplary aralkyloxyl group is benzyloxyl,i.e., C₆H₅—CH₂—O—. An aralkyloxyl group can optionally be substituted.

“Alkoxycarbonyl” refers to an alkyl-O—C(═O)— group. Exemplaryalkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl,butyloxycarbonyl, and tert-butyloxycarbonyl.

“Aryloxycarbonyl” refers to an aryl-O—C(═O)— group. Exemplaryaryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.

“Aralkoxycarbonyl” refers to an aralkyl-O—C(═O)— group. An exemplaryaralkoxycarbonyl group is benzyloxycarbonyl.

“Carbamoyl” refers to an amide group of the formula —C(═O)NH₂.“Alkylcarbamoyl” refers to a R′RN—C(═O)— group wherein one of R and R′is hydrogen and the other of R and R′ is alkyl and/or substituted alkylas previously described. “Dialkylcarbamoyl” refers to a R′RN—C(═O)—group wherein each of R and R′ is independently alkyl and/or substitutedalkyl as previously described.

The term carbonyldioxyl, as used herein, refers to a carbonate group ofthe formula —O—C(═O)—OR.

“Acyloxyl” refers to an acyl-O— group wherein acyl is as previouslydescribed.

The term “amino” refers to the —NH₂ group and also refers to a nitrogencontaining group as is known in the art derived from ammonia by thereplacement of one or more hydrogen radicals by organic radicals. Forexample, the terms “acylamino” and “alkylamino” refer to specificN-substituted organic radicals with acyl and alkyl substituent groupsrespectively.

An “aminoalkyl” as used herein refers to an amino group covalently boundto an alkylene linker. More particularly, the terms alkylamino,dialkylamino, and trialkylamino as used herein refer to one, two, orthree, respectively, alkyl groups, as previously defined, attached tothe parent molecular moiety through a nitrogen atom. The term alkylaminorefers to a group having the structure —NHR′ wherein R′ is an alkylgroup, as previously defined; whereas the term dialkylamino refers to agroup having the structure —NR′R″, wherein R′ and R″ are eachindependently selected from the group consisting of alkyl groups. Theterm trialkylamino refers to a group having the structure —NR′R″R′″,wherein R′, R″, and R′″ are each independently selected from the groupconsisting of alkyl groups. Additionally, R′, R″, and/or R′″ takentogether may optionally be —(CH₂)_(k)— where k is an integer from 2 to6. Examples include, but are not limited to, methylamino, dimethylamino,ethylamino, diethylamino, diethylaminocarbonyl, methylethylamino,isopropylamino, piperidino, trimethylamino, and propylamino.

The amino group is —NR′R″, wherein R′ and R″ are typically selected fromhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl.

The terms alkylthioether and thioalkoxyl refer to a saturated (i.e.,alkyl-S—) or unsaturated (i.e., alkenyl-S— and alkynyl-S—) groupattached to the parent molecular moiety through a sulfur atom. Examplesof thioalkoxyl moieties include, but are not limited to, methylthio,ethylthio, propylthio, isopropylthio, n-butylthio, and the like.

“Acylamino” refers to an acyl-NH— group wherein acyl is as previouslydescribed. “Aroylamino” refers to an aroyl-NH— group wherein aroyl is aspreviously described.

The term “carbonyl” refers to the —C(═O)— group, and can include analdehyde group represented by the general formula R—C(═O)H.

The term “carboxyl” refers to the —COOH group. Such groups also arereferred to herein as a “carboxylic acid” moiety.

The terms “halo,” “halide,” or “halogen” as used herein refer to fluoro,chloro, bromo, and iodo groups. Additionally, terms such as “haloalkyl,”are meant to include monohaloalkyl and polyhaloalkyl. For example, theterm “halo(C₁-C₄)alkyl” is mean to include, but not be limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, andthe like.

The term “hydroxyl” refers to the —OH group.

The term “hydroxyalkyl” refers to an alkyl group substituted with an —OHgroup.

The term “mercapto” refers to the —SH group.

The term “oxo” as used herein means an oxygen atom that is double bondedto a carbon atom or to another element.

The term “nitro” refers to the —NO₂ group.

The term “thio” refers to a compound described previously herein whereina carbon or oxygen atom is replaced by a sulfur atom.

The term “sulfate” refers to the —SO₄ group.

The term thiohydroxyl or thiol, as used herein, refers to a group of theformula —SH.

More particularly, the term “sulfide” refers to compound having a groupof the formula —SR

The term “sulfone” refers to compound having a sulfonyl group —S(O₂)R.

The term “sulfoxide” refers to a compound having a sulfinyl group —S(O)R

The term ureido refers to a urea group of the formula —NH—CO—NH₂.

Throughout the specification and claims, a given chemical formula orname shall encompass all tautomers, congeners, and optical- andstereoisomers, as well as racemic mixtures where such isomers andmixtures exist.

Certain compounds of the present disclosure may possess asymmetriccarbon atoms (optical or chiral centers) or double bonds; theenantiomers, racemates, diastereomers, tautomers, geometric isomers,stereoisometric forms that may be defined, in terms of absolutestereochemistry, as (R)- or (S)- or, as D- or L- for amino acids, andindividual isomers are encompassed within the scope of the presentdisclosure. The compounds of the present disclosure do not include thosewhich are known in art to be too unstable to synthesize and/or isolate.The present disclosure is meant to include compounds in racemic,scalemic, and optically pure forms. Optically active (R)- and (S)-, orD- and L-isomers may be prepared using chiral synthons or chiralreagents, or resolved using conventional techniques. When the compoundsdescribed herein contain olefenic bonds or other centers of geometricasymmetry, and unless specified otherwise, it is intended that thecompounds include both E and Z geometric isomers. Unless otherwisestated, structures depicted herein are also meant to include allstereochemical forms of the structure; i.e., the R and S configurationsfor each asymmetric center. Therefore, single stereochemical isomers aswell as enantiomeric and diastereomeric mixtures of the presentcompounds are within the scope of the disclosure.

It will be apparent to one skilled in the art that certain compounds ofthis disclosure may exist in tautomeric forms, all such tautomeric formsof the compounds being within the scope of the disclosure. The term“tautomer,” as used herein, refers to one of two or more structuralisomers which exist in equilibrium and which are readily converted fromone isomeric form to another.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures with the replacement of a hydrogen by a deuterium or tritium,or the replacement of a carbon by ¹³C- or ¹⁴C-enriched carbon are withinthe scope of this disclosure.

The compounds of the present disclosure may also contain unnaturalproportions of atomic isotopes at one or more of atoms that constitutesuch compounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example iodine-125 (¹²⁵I) orastatine-211 (²¹¹At). All isotopic variations of the compounds of thepresent disclosure, whether radioactive or not, are encompassed withinthe scope of the present disclosure.

The compounds of the present disclosure may exist as salts. The presentdisclosure includes such salts. Examples of applicable salt formsinclude hydrochlorides, hydrobromides, sulfates, methanesulfonates,nitrates, maleates, acetates, citrates, fumarates, tartrates (e.g.(+)-tartrates, (−)-tartrates or mixtures thereof including racemicmixtures, succinates, benzoates and salts with amino acids such asglutamic acid. These salts may be prepared by methods known to thoseskilled in art. Also included are base addition salts such as sodium,potassium, calcium, ammonium, organic amino, or magnesium salt, or asimilar salt. When compounds of the present disclosure containrelatively basic functionalities, acid addition salts can be obtained bycontacting the neutral form of such compounds with a sufficient amountof the desired acid, either neat or in a suitable inert solvent or byion exchange. Examples of acceptable acid addition salts include thosederived from inorganic acids like hydrochloric, hydrobromic, nitric,carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfiric, hydriodic, orphosphorous acids and the like, as well as the salts derived organicacids like acetic, propionic, isobutyric, maleic, malonic, benzoic,succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike. Certain specific compounds of the present disclosure contain bothbasic and acidic functionalities that allow the compounds to beconverted into either base or acid addition salts.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents.

Certain compounds of the present disclosure can exist in unsolvatedforms as well as solvated forms, including hydrated forms. In general,the solvated forms are equivalent to unsolvated forms and areencompassed within the scope of the present disclosure. Certaincompounds of the present disclosure may exist in multiple crystalline oramorphous forms. In general, all physical forms are equivalent for theuses contemplated by the present disclosure and are intended to bewithin the scope of the present disclosure.

In addition to salt forms, the present disclosure provides compounds,which are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentdisclosure. Additionally, prodrugs can be converted to the compounds ofthe present disclosure by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present disclosure when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

The term “protecting group” refers to chemical moieties that block someor all reactive moieties of a compound and prevent such moieties fromparticipating in chemical reactions until the protective group isremoved, for example, those moieties listed and described in T. W.Greene, P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd ed.John Wiley & Sons (1999). It may be advantageous, where differentprotecting groups are employed, that each (different) protective groupbe removable by a different means. Protective groups that are cleavedunder totally disparate reaction conditions allow differential removalof such protecting groups. For example, protective groups can be removedby acid, base, and hydrogenolysis. Groups such as trityl,dimethoxytrityl, acetal and tert-butyldimethylsilyl are acid labile andmay be used to protect carboxy and hydroxy reactive moieties in thepresence of amino groups protected with Cbz groups, which are removableby hydrogenolysis, and Fmoc groups, which are base labile. Carboxylicacid and hydroxy reactive moieties may be blocked with base labilegroups such as, without limitation, methyl, ethyl, and acetyl in thepresence of amines blocked with acid labile groups such as tert-butylcarbamate or with carbamates that are both acid and base stable buthydrolytically removable.

Carboxylic acid and hydroxy reactive moieties may also be blocked withhydrolytically removable protective groups such as the benzyl group,while amine groups capable of hydrogen bonding with acids may be blockedwith base labile groups such as Fmoc. Carboxylic acid reactive moietiesmay be blocked with oxidatively-removable protective groups such as2,4-dimethoxybenzyl, while co-existing amino groups may be blocked withfluoride labile silyl carbamates.

Allyl blocking groups are useful in the presence of acid- andbase-protecting groups since the former are stable and can besubsequently removed by metal or pi-acid catalysts. For example, anallyl-blocked carboxylic acid can be deprotected with a palladium(O)—catalyzed reaction in the presence of acid labile t-butyl carbamate orbase-labile acetate amine protecting groups. Yet another form ofprotecting group is a resin to which a compound or intermediate may beattached. As long as the residue is attached to the resin, thatfunctional group is blocked and cannot react. Once released from theresin, the functional group is available to react.

Typical blocking/protecting groups include, but are not limited to thefollowing moieties:

Further, as used herein, a “protecting group” is a chemical substituentwhich can be selectively removed by readily available reagents which donot attack the regenerated functional group or other functional groupsin the molecule. Suitable protecting groups are known in the art andcontinue to be developed. Suitable protecting groups may be found, forexample in Wutz et al. (“Greene's Protective Groups in OrganicSynthesis, Fourth Edition,” Wiley-Interscience, 2007). Protecting groupsfor protection of the carboxyl group, as described by Wutz et al. (pages533-643), are used in certain embodiments. In some embodiments, theprotecting group is removable by treatment with acid. Specific examplesof protecting groups include, but are not limited to, benzyl,p-methoxybenzyl (PMB), tertiary butyl (t-Bu), methoxymethyl (MOM),methoxyethoxymethyl (MEM), methylthiomethyl (MTM), tetrahydropyranyl(THP), tetrahydrofuranyl (THF), benzyloxymethyl (BOM), trimethylsilyl(TMS), triethylsilyl (TES), t-butyldimethylsilyl (TBDMS), andtriphenylmethyl (trityl, Tr). Persons skilled in the art will recognizeappropriate situations in which protecting groups are required and willbe able to select an appropriate protecting group for use in aparticular circumstance.

The term “metal ion” as used herein refers to elements of the periodictable that are metallic and that are positively charged as a result ofhaving fewer electrons in the valence shell than is present for theneutral metallic element. Metals that are useful in the presentlydisclosed subject matter include metals capable of formingpharmaceutically acceptable compositions. Useful metals include, but arenot limited to, Li, Na, K, Rb, Cs, Be, Mg, Ca, Sr, and Ba. One of skillin the art will appreciate that the metals described above can eachadopt several different oxidation states. In some instances, the moststable oxidation state is formed, but other oxidation states are usefulin the presently disclosed subject matter.

Following long-standing patent law convention, the terms “a,” “an,” and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a subject” includes aplurality of subjects, unless the context clearly is to the contrary(e.g., a plurality of subjects), and so forth.

Throughout this specification and the claims, the terms “comprise,”“comprises,” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise. Likewise, the term “include” andits grammatical variants are intended to be non-limiting, such thatrecitation of items in a list is not to the exclusion of other likeitems that can be substituted or added to the listed items.

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing amounts, sizes, dimensions,proportions, shapes, formulations, parameters, percentages, quantities,characteristics, and other numerical values used in the specificationand claims, are to be understood as being modified in all instances bythe term “about” even though the term “about” may not expressly appearwith the value, amount or range. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the followingspecification and attached claims are not and need not be exact, but maybe approximate and/or larger or smaller as desired, reflectingtolerances, conversion factors, rounding off, measurement error and thelike, and other factors known to those of skill in the art depending onthe desired properties sought to be obtained by the presently disclosedsubject matter. For example, the term “about,” when referring to a valuecan be meant to encompass variations of, in some embodiments, ±100% insome embodiments ±50%, in some embodiments ±20%, in some embodiments±10%, in some embodiments ±5%, in some embodiments ±1%, in someembodiments ±0.5%, and in some embodiments f 0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethods or employ the disclosed compositions.

Further, the term “about” when used in connection with one or morenumbers or numerical ranges, should be understood to refer to all suchnumbers, including all numbers in a range and modifies that range byextending the boundaries above and below the numerical values set forth.The recitation of numerical ranges by endpoints includes all numbers,e.g., whole integers, including fractions thereof, subsumed within thatrange (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5,as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like)and any range within that range.

In the examples below the following terms are intended to have thefollowing meaning: ACN: acetonitrile, DCM: Dichloromethane, DIPEA:N,N-Diisopropylethylamine, DMF: Dimethylformamide, HPLC: HighPerformance Liquid Chromatography, HRMS: High Resolution MassSpectrometry, LRMS: Low Resolution Mass Spectrometry, NCS:N-Chlorosuccinimide, NHS: N-Hydroxysuccinimide, NMR: nuclear magneticresonance, PMB: p-methoxybenzyl, RT: room temperature, TEA:Triethylamine, TFA: Trifluoroacetic acid, and TSTU:O—(N-Succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate.

EXAMPLES

The following Examples have been included to provide guidance to one ofordinary skill in the art for practicing representative embodiments ofthe presently disclosed subject matter. In light of the presentdisclosure and the general level of skill in the art, those of skill canappreciate that the following Examples are intended to be exemplary onlyand that numerous changes, modifications, and alterations can beemployed without departing from the scope of the presently disclosedsubject matter. The synthetic descriptions and specific examples thatfollow are only intended for the purposes of illustration, and are notto be construed as limiting in any manner to make compounds of thedisclosure by other methods.

Example 1 General Methods

Chemistry. All chemicals and solvents were purchased from eitherSigma-Aldrich (Milwaukee, Wis.) or Fisher Scientific (Pittsburgh, Pa.).The N-hydroxysuccinimide (NHS) esters of DyLight 800 was purchased fromThermo Fisher Scientific (Rockford, Ill.). ESI mass spectra wereobtained on a Bruker Esquire 3000 plus system (Billerica, Mass.).High-performance liquid chromatography (HPLC) purifications wereperformed on a Varian Prostar System (Varian Medical Systems, Palo Alto,Calif.).

Cell Lines and Tumor Models. PSMA⁺ PC3 PIP and PSMA− PC3 flu cell lineswere obtained from Dr. Warren Heston (Cleveland Clinic). Cells weregrown to 80-90% confluence in a single passage before trypsinization andformulation in Hank's balanced salt solution (HBSS, Sigma, St. Louis,Mo.) for implantation into mice. Animal studies were carried out incompliance with guidelines related to the conduct of animal experimentsof the Johns Hopkins Animal Care and Use Committee. For optical imagingstudies and ex-vivo biodistribution, male NOD-SCID mice (JHU, in housecolony) were implanted subcutaneously with 1×10⁶ PSMA⁺ PC3 PIP and PSMA⁻PC3 flu cells in opposite flanks. Mice were imaged when the tumorxenografts reached 3-5 mm in diameter.

In Vivo Imaging and E Vivo Biodistribution. After image acquisition atbaseline (pre-injection), mouse was injected intravenously with 1 nmolof DyLight800-3 and images were acquired at 1h, 2h, 4h and 24h timepoints using a Pearl Impulse Imager (LI-COR Biosciences). Following the24 h image the mouse was sacrificed by cervical dislocation and tumor,muscle, liver, spleen, kidneys and intestine were collected andassembled on a petri dish for image acquisition. All images were scaledto the same intensity for direct comparison. FIG. 1 shows the images at24 hours postinjection of 1 nmol of DyLight800-3 in mouse with PSMA+PC3PIP and PSMA− PC3 flu tumors. Both whole body and ex vivo organ imagingclearly demonstrated PSMA+ PC3 PIP tumor uptake and little uptake inPSMA− PC3 flu tumor, indicating target selectivity in vivo.

Example 2 Synthesis Methods

Synthesis of DyLight800-3: To a solution of compound 3, Chen et al.,2009, (0.5 mg, 0.7 μmol) in DMSO (0.1 mL) was addedN,N-diisopropylethylamine (0.010 mL, 57.4 μmol), followed by NHS esterof DyLight800 (0.3 mg, 0.29 μmol). After 1 h at room temperature, thereaction mixture was purified by HPLC (column, Phenomenex Luna C18 10μ,250×4.6 mm; mobile phase, A=0.1% TFA in H₂O, B=0.1% TFA in CH₃CN;gradient, 0 min=5% B, 5 min=5% B, 45 min=100% B; flow rate, 1 mL/min) toafford 0.3 mg (70%) of DyLight800-3: ESI-Mass calcd for C₇₁H₉₄N₇O₂₂S₃ ⁻[M−H]⁻ 1492.6, found 1492.4 [M−H]⁻.

Example 3 General

All reagents and solvents were purchased from either Sigma-Aldrich(Milwaukee, Wis.) or Fisher Scientific (Pittsburgh, Pa.).2-{3-[5-[7-(2,5-Dioxo-pyrrolidin-1-yloxycarbonyl)-heptanoylamino]-1-(4-methoxy-benzyloxycarbonyl)-pentyl]-ureido}-pentanedioicacid bis-(4-methoxy-benzyl) ester (1) was prepared according to(Banerjee et al., J. Med. Chem., vol. 51, pp. 4504-4517, 2008).H-Lys(Boc)-OBu.HCl was purchased from Chem-Impex International (WoodDale, Ill.). The N-hydroxysuccinimide (NHS) ester of IRDye 800CW waspurchased from LI-COR Biosciences (Lincoln, Nebr.). ¹H NMR spectra wereobtained on a Bruker Avance 400 mHz Spectrometer. ESI mass spectra wereobtained on a Bruker Esquire 3000 plus system. Purification byhigh-performance liquid chromatography (HPLC) was performed on a VarianProstar System (Varian Medical Systems, Palo Alto, Calif.). YC-27

Compound YC-27 was prepared according the scheme shown below.

Trifluoroacetate salt of2-(3-{5-[7-(5-amino-1-carboxy-pentylcarbamoyl)-heptanoylamino]-1-carboxy-pentyl}-ureido)-pentanedioicacid (YC-VIII-24). To a solution of 1 (0.065 g, 0.020 mmol) in CH₂Cl₂ (2mL) was added triethylamine (0.040 mL, 0.285 mmol), followed byH-Lys(Boc)-OBu.HCl (0.028 g, 0.083 mmol). After stirring for 2 h at roomtemperature, the solvent was evaporated on a rotary evaporator. Asolution of TFA/CH₂Cl₂ 1:1 (2 mL) was then added to the residue andstirred for 1 h at room temperature. The crude material was purified byHPLC (column, Econosphere C18, 10μ, 250×10 mm; retention time, 15 min;mobile phase, A=0.1% TFA in H₂O, B=0.1% TFA in CH₃CN; gradient, 0 min=5%B, 25 min=25% B; flow rate, 4 mL/min) to afford 0.032 g (66%) ofYC-VIII-24. ¹H NMR (400 MHz, D₂O) δ 4.24-4.28 (m, 1H), 4.17-4.20 (m,1H), 4.08-4.12 (m, 1H), 3.08-3.12 (m, 2H), 2.88-2.92 (m, 2H), 2.41-2.44(m, 2H), 2.19-2.21 (m, 2H), 2.05-2.16 (m, 3H), 1.57-1.93 (m, 7H),1.21-1.50 (m, 10H), 1.21 (m, 4H). ESI-Mass calcd for C₂₆H₄N₅O₁₁ [M]⁺604.3, found 604.0.

YC-27. To a solution of YC-VIII-24 (0.3 mg, 0.43 μmol) in DMSO (0.1 mL)was added N,N-diisopropylethylamine (0.002 mL, 11.4 μmol), followed bythe NHS ester of IRDye 800CW (0.3 mg, 0.26 μmol). After stirring forYC-VIII-24 for 2 h at room temperature, the reaction mixture waspurified by HPLC (column, Econosphere C18 5μ, 150×4.6 mm; retentiontime, 22 min, mobile phase, A=0.1% TFA in H₂O, B=0.1% TFA in CH₃CN;gradient, 0 min=0% B, 5 min=0% B, 45 min=100% B; flow rate, 1 mL/min) toafford 0.3 mg (72%) of YC-27. ESI-Mass calcd for C₇₂H₉₇N₇O₂₅S₄ [M]⁺1587.5, found 794.3 [M+H]²⁺, 1587.6 [M]⁺.

Synthesis of Precursor YC-VI-54

To a solution of Lys-Urea-Glu (0.103 g, 0.121 mmol, Banerjee et al J.Med. Chem., vol. 51, pp. 4507-4517, 2008) in DMF (2 mL) was addedBoc-NH-PEG-COOH (0.060 g, 0.135 mmol) and TBTU (0.040 g, 0.125 mmol),followed by N,N′-diisopropylethylamine (0.042 mL, 0.241 mmol). Afterstirring overnight at room temperature, the solvent was evaporated on arotary evaporator. The crude material was purified by a silica columnusing methanol/methylene chloride (5:95) to afford 0.101 g (0.109 mmol,90%) of YC-VI-53, which was dissolved in a solution of 3% anisole in TFA(1 mL). The mixture was reacted at room temperature for 10 min, thenconcentrated on a rotary evaporator. The crude material was purified byHPLC (Econosphere C18 10u, 250×10 mm, H₂O/CH₃CN/TFA (92/8/0.1), 4mL/min, Compound YC-VI-54 eluting at 11 min) to afford 0.035 g (57%) ofcompound YC-VI-54. ¹H NMR (400 MHz, D₂O) δ 4.17-4.21 (m, 1H), 4.10-4.13(m, 1H), 4.00 (s, 2H), 3.67-3.71 (m, 6H), 3.14-3.20 (m, 4H), 2.43-2.46(m, 2H), 2.08-2.13 (m, 1H), 1.87-1.93 (m, 1H), 1.76-1.79 (m, 1H),1.63-1.67 (m, 1H), 1.45-1.50 (m, 2H), 1.33-1.40 (m, 2H). ESI-Mass calcdfor C₁₈H₃₃N₄O₁₀ [M]⁺ 465.2, found 465.2.

To a solution of compound YC-VI-54 (0.3 mg, 53 μmol) in DMSO (0.05 mL)was added N,N-diisopropylethylamine (0.002 mL, 11.4 μmol), followed byNHS ester of IRDye 800RS (0.2 mg, 0.21 μmol). After 2 hour at roomtemperature, the reaction mixture was purified by HPLC (column,Econosphere C18 5μ, 150×4.6 mm; retention time, 28 min; mobile phase,A=0.1% TFA in H₂O, B=0.1% TFA in CH₃CN; gradient, 0 mins=0% B, 5 mins=0%B, 45 mins=100% B; flow rate, 1 mL/min) to afford 0.2 mg (75%) ofcompound YC-VIII-11. ESI-Mass calcd for C₆₄H₈₄N₆O₁₈S₂ [M]⁺ 1288.5, found1288.9.

To a solution of compound YC-VI-54 (0.3 mg, 53 μmol) in DMSO (0.05 mL)was added N,N-diisopropylethylamine (0.002 mL, 11.4 μmol), followed byNHS ester of IRDye800CW (0.2 mg, 0.17 μmol). After 2 hour at roomtemperature, the reaction mixture was purified by HPLC (column,Econosphere C18 5μ, 150×4.6 mm; retention time, 22 min; mobile phase,A=0.1% TFA in H₂O, B=0.1% TFA in CH₃CN; gradient, 0 mins=0% B, 5 mins=0%B, 45 mins=100% B; flow rate, 1 mL/min) to afford 0.2 mg (80%) ofcompound YC-VIII-12. ESI-Mass calcd for C₆₄H₈₄N₆O₂₄S₄ [M]⁺ 1448.4, found1448.7.

To a solution of YC-VIII-24 (prepared as described previously for YC-27)(0.3 mg, 0.42 μmol) in DMSO (0.1 mL) was added N,N-diisopropylethylamine(0.002 mL, 11.5 μmol), followed by NHS ester of IRDye 800RS (0.3 mg,0.31 μmol). After 2 hour at room temperature, the reaction mixture waspurified by HPLC (column, Econosphere C18 5μ, 150×4.6 mm; retentiontime, 27 min; mobile phase, A=0.1% TFA in H₂O, B=0.1% TFA in CH₃CN;gradient, 0 mins=0% B, 5 mins=0% B, 45 mins=100% B; flow rate, 1 mL/min)to afford 0.3 mg (67%) of compound YC-VIII-28. ESI-Mass calcd forC₇₂H₉₇N₇O₁₉S₂ [M]⁺ 1427.6, found 714.4 [M+H]²⁺, 1427.8 [M]⁺.

To a solution of YC-VIII-24 (0.5 mg, 0.70 μmol) in DMSO (0.1 mL) wasadded N,N-diisopropylethylamine (0.005 mL, 28.7 μmol), followed by NHSester of BODIPY 650/665-X (0.3 mg, 0.47 μmol). After 2 hour at roomtemperature, the reaction mixture was purified by HPLC (column,Econosphere C18 5μ, 150×4.6 mm; retention time, 28 min; mobile phase,A=0.1% TFA in H₂O, B=0.1% TFA in CH₃CN; gradient, 0 mins=0% B, 5 mins=0%B, 45 mins=100% B; flow rate, 1 mL/min) to afford 0.4 mg (75%) ofcompound YC-VIII-30. ESI-Mass calcd for C₅₅H₇₃BF₂N₉O₄ [M+H]⁺ 1132.5,found 1132.0.

To a solution of YC-VI-54 (0.5 mg, 0.70 μmol) in DMSO (0.1 mL) was addedN,N-diisopropylethylamine (0.005 mL, 28.7 μmol), followed by NHS esterof BODIPY 650/665-X (0.3 mg, 0.47 μmol). After 2 hour at roomtemperature, the reaction mixture was purified by HPLC (column,Econosphere C18 5μ, 150×4.6 mm; retention time, 29 min; mobile phase,A=0.1% TFA in H₂O, B=0.1% TFA in CH₃CN; gradient, 0 mins=0% B, 5 mins=0%B, 45 mins=100% B; flow rate, 1 mL/min) to afford 0.4 mg (86%) ofcompound YC-VIII-31. ESI-Mass calcd for C₄₇H₅₉BF₂N₈O₁₃ [M]⁺ 992.4, found992.9.

To a solution of Lys-Urea-Glu (4.0 mg, 9.6 μmol) in DMF (0.5 mL) wasadded triethylamine (0.01 mL, 71.7 μmol), followed by Marina Blue-NHSester (1.8 mg, 4.9 μmol). After 2 hour at room temperature, the reactionmixture was purified by HPLC (column, Econosphere C18 10μ, 250×10 mm;retention time, 14 min; mobile phase, H₂O/CH₃CN/TFA=85/15/0.1; flowrate, 4 mL/min) to afford 2.5 mg (89%) of compound YC-VIII-41. ¹H NMR(400 MHz, D₂O) δ 7.40 (d, J=11.6 Hz, 1H), 4.23-4.31 (m, 1H), 4.15-4.19(m, 1H), 3.64 (s, 2H), 3.19-3.23 (m, 2H), 2.49-2.53 (m, 2H), 2.39 (s,3H), 2.06-2.17 (m, 1H), 1.95-1.99 (m, 1H), 1.83-1.90 (m, 1H), 1.72-1.80(m, 1H), 1.52-1.55 (m, 2H), 1.40-1.45 (m, 2H). ESI-Mass calcd forC₂₄H₂₈F₂N₃O₁₁ [M+H]⁺ 572.2, found 571.8.

To a solution of Lys-Urea-Glu (4.0 mg, 9.6 μmol) in DMSO (0.5 mL) wasadded N,N-diisopropylethylamine (0.020 mL, 114.8 μmol), followed by4-[2-(4-dimethylamino-phenyl)-vinyl]-1-(3-isothiocyanato-propyl)-pyridium(3 mg, 7.4 μmol). After 2 hour at room temperature, the reaction mixturewas purified by HPLC (column, Econosphere C18 10μ, 250×10 mm; retentiontime, 13 min; mobile phase, A=0.1% TFA in H₂O, B=0.1% TFA in CH₃CN;gradient, 0 mins=10% B, 20 mins=60% B; flow rate, 4 mL/min) to afford1.3 mg (24%) of compound YC-VIII-52. ESI-Mass calcd for C₃₁H₄₃N₆O₇S [M]⁺643.3, found 642.9.

To a solution of YC-VIII-24 (3.0 mg, 4.2 μmol) in DMSO (0.5 mL) wasadded N,N-diisopropylethylamine (0.020 mL, 114.8 μmol), followed by4-[2-(4-dimethylamino-phenyl)-vinyl]-1-(3-isothiocyanato-propyl)-pyridium(2 mg, 4.9 μmol). After 2 hour at room temperature, the reaction mixturewas purified by HPLC (column, Econosphere C18 5μ, 150×4.6 mm; retentiontime, 15 min; mobile phase, A=0.1% TFA in H₂O, B=0.1% TFA in CH₃CN;gradient, 0 mins=0% B, 5 mins=0% B, 45 mins=100% B; flow rate, 1 mL/min)to afford 2 mg (47%) of compound YC-VIII-74. ESI-Mass calcd forC₄₅H₆₇N₈O₁₁S [M]⁺ 927.5, found 927.0.

To a solution of YC-VIII-24 (5.0 mg, 7.0 μmol) in DMF (1 ML) was addedtriethylamine (0.020 mL, 143.5 μmol), followed by NHS ester of5-(and-6)-carboxynaphthofluorescein (4.0 mg, 7.0 μmol). After 1 hour atroom temperature, the reaction mixture was purified by HPLC (column,Econosphere C18 10μ, 250×10 mm; retention time, minor product at 17 min,major product at 20 min); mobile phase, H₂O/CH₃CN/TFA=70/30/0.1; flowrate, 4 mL/min) to afford 0.3 mg of minor and 2.2 mg of major product(two isomers of YC-VIII-63). ESI-Mass calcd for C₅₅H₅₉N₅O₁₇ [M]⁺ 1061.4,found 1061.6 (for both minor and major product).

To a solution of Lys-Urea-Glu (0.2 mg, 0.48 μmol) in DMSO (0.05 mL) wasadded N,N-diisopropylethylamine (0.002 mL, 11.5 μmol), followed by NHSester of IRDye 800RS (0.2 mg, 0.21 μmol). After 2 hour at roomtemperature, the reaction mixture was purified by HPLC (column,Econosphere C18 5μ, 150×4.6 mm; retention time, 23 min; mobile phase,A=0.1% TFA in H₂O, B=0.1% TFA in CH₃CN; gradient, 0 mins=0% B, 5 mins=0%B, 45 mins=100% B; flow rate, 1 mL/min) to afford 0.2 mg (84%) ofcompound YC-IX-92. ESI-Mass calcd for C₅₈H₇₃N₅O₁₅S₂ [M]⁺ 1143.5, found572.5 [M+H]²⁺, 1144.0 [M]⁺.

Characterization —Fluorescence

Fluorescence spectra were recorded using a Varian Cary Eclipsefluorescence spectrophotometer (Varian Medical Systems) with excitationfrom a Xenon arc lamp. YC-27 was dissolved in water. All of thefluorescence measurements were performed in aqueous solution underambient conditions. The fluorescence quantum yield of YC-27 was measuredusing an aqueous solution of ICG (Φ=0.016 (Sevick-Muraca et al.,Photochem. Photobiol., vol. 66, pp. 55-64, 1997), excitation wavelengthat 775 nm) as the standard (FIG. 2). The fluorescence intensity datawere collected in the spectral region 780-900 nm over which quantumyield was integrated. Time-resolved intensity decays were recorded usinga PicoQuant Fluotime 100 time-correlated single-photon counting (TCSPC)fluorescence lifetime spectrometer (PicoQuant, Berlin, Del.). Theexcitation was obtained using a pulsed laser diode (PicoQuant PDL800-B)with a 20 MHz repetition rate. The fluorescence intensity decay of YC-27was analyzed in terms of the single-exponential decay using thePicoQuant Fluofit 4.1 software with deconvolution of the instrumentresponse function and nonlinear least squares fitting. Thegoodness-of-fit was determined by the χ² value.

The electronic spectrum of YC-27 exhibited an absorbance maximum at 774nm with an extinction coefficient of 158,900 M⁻¹. Upon excitation, YC-27provided intense fluorescence with an emission maximum at 792 nm and afluorescence lifetime of 443 psec in aqueous solution (FIG. 3). Using anexcitation wavelength of 775 nm, YC-27 demonstrated a fluorescencequantum yield of 0.053 in aqueous solution relative to ICG, whichdemonstrated a quantum yield of 0.016 (FIG. 2) (Sevick-Muraca et al.,Photochem. Photobiol., vol. 66, pp. 55-64, 1997), attesting to theefficiency of this IRDye 800CW-based compound. That is significantbecause ICG has been used previously for intraoperative tumor mapping(K. Gotoh, T. Yamada, O. Ishikawa, H. Takahashi, H. Eguchi, M. Yano, H.Ohigashi, Y. Tomita, Y. Miyamoto, and S. Imaoka, A novel image-guidedsurgery of hepatocellular carcinoma by indocyanine green fluorescenceimaging navigation. J. Surg. Oncol., 2009).

In Vitro NAALADase Activity

PSMA inhibitory activity of YC-27 was determined using afluorescence-based assay according to a previously reported procedure(Chen et al., J. Med. Chem., vol. 51, pp. 7933-7943, 2008). Briefly,lysates of LNCaP cell extracts (25 μL) were incubated with the inhibitor(12.5 μL) in the presence of 4 μM N-acetylaspartylglutamate (NAAG) (12.5μL) for 120 min. The amount of glutamate released by NAAG hydrolysis wasmeasured by incubation with a working solution (50 μL) of the Amplex RedGlutamic Acid Kit (Molecular Probes Inc., Eugene, Oreg.) for 60 min.Fluorescence was measured with a VICTOR³V multilabel plate reader(Perkin Elmer Inc., Waltham, Mass.) with excitation at 530 nm andemission at 560 nm. Inhibition curves were determined using semi-logplots, and IC₅₀ values were determined at the concentration at whichenzyme activity was inhibited by 50%. Assays were performed intriplicate. Enzyme inhibitory constants (K_(i) values) were generatedusing the Cheng-Prusoff conversion. Data analysis was performed usingGraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego,Calif.).

This assay is free from the interference of IRDye 800CW because theexcitation/emission maxima of IRDye 800CW are remote from those ofresorufin (λ_(ex)=563 nm, λ_(em)=587 nm), which provides the fluorescentreadout in the assay. The K_(i) value of YC-27 was 0.37 nM with 95%confidence intervals from 0.18 nM to 0.79 nM. Under the sameexperimental conditions, the K_(i) value of the known PSMA inhibitorZJ-43 (Zhou et al., Nat. Rev. Drug Discov., vol. 4, pp. 1015-1026, 2005)was 2.1 nM, indicating the high inhibitory capacity of YC-27. Theinhibition curve of YC-27, which is expressed with respect to the amountof glutamate released from hydrolysis of NAAG, is shown in FIG. 4.

Biodistribution and Imaging

Cell Culture and Animal Models. Both PSMA-expressing (PSMA+ PC3− PIP)and non-expressing (PSMA− PC3-flu) prostate cancer cell lines (Chang etal, Cancer Res., vol. 59, pp. 3192-3198, 1999) were grown in RPMI 1640medium (Invitrogen, Carlsbad, Calif.) containing 10% fetal bovine serum(FBS) (Invitrogen) and 1% Pen-Strep (Biofluids, Camarillo, Calif.). Allcell cultures were maintained in 5% carbon dioxide (CO₂), at 37.0° C. ina humidified incubator. Animal studies were undertaken in compliancewith the regulations of the Johns Hopkins Animal Care and Use Committee.Six- to eight-week-old male, non-obese diabetic (NOD)/severe-combinedimmunodeficient (SCID) mice (Charles River Laboratories, Wilmington,Mass.) were implanted subcutaneously (s.c.) with PC3-PIP and PC3-flucells (2×10⁶ in 100 □L of Matrigel) at the forward left and rightflanks, respectively. Mice were imaged or used in ex vivobiodistribution assays when the xenografts reached 5 to 7 mm indiameter.

In vivo Imaging and Ex vivo Biodistribution. Mouse #1 was injected with10 nmol and mouse #2 with 1 nmol of YC-27 in 200 μL of PBS intravenously(i.v.) via the lateral tail vein. Mouse #3 was injected with 1 nmol ofYC-27 and also co-injected with 1 □mol of the known PSMA inhibitor2-{3-[1-carboxy-5-(4-iodo-benzoylamino)-pentyl]-ureido}-pentanedioicacid (DCIBzL) (Chen et al., J. Med. Chem., vol. 51, pp. 7933-7943, 2008;Barinka et al., J. Med. Chem. vol. 51, pp. 7737-7743, 2008) in 200 μL ofPBS i.v. to assess for PSMA binding specificity. Images were acquired atan array of post-injection (p.i.) time points starting at 10 min p.i.using a dedicated small animal optical imaging instrument, the PearlImager (LI-COR Biosciences). The Pearl Imager uses diffusive lasersoptimized for IRDye 800CW. The instrument employs a CCD camera with afield-of-view of 11.2 cm×8.4 cm at the surface of the imaging bed. Thescan time was less than 30 sec to complete white light, 700 nm channeland 800 nm channel image acquisition. Images are displayed using apseudocolor output with corresponding scale. All images were acquired atthe same parameter settings and are scaled to the same maximum values.Imaging bed temperature was adjusted to 37° C. Animals receivedinhalational anesthesia (isoflurane) through a nose cone attached to theimaging bed. Animals were sacrificed by cervical dislocation for ex vivoimaging studies at the end of acquisition of the in vivo images. Ex vivoimages were acquired first by midline surgical laparotomy and then againby harvesting liver, spleen, stomach, small intestine, kidneys, urinarybladder, PC3-PIP and PC3-flu tumors and displaying them individually onplastic Petri dishes. Estimates of signal output were provided bydrawing three circular regions of interest within each tumor anddetermining the average signal (arbitrary units)/area using themanufacturer's software.

FIG. 5A-FIG. 5O (mouse #1) depict the pharmacokinetic behavior of YC-27in vivo. In this experiment 10 nmol of YC-27 was administeredintravenously and the animal was imaged repeatedly over a three dayperiod. Although difficult to quantify as these are planar images, onecan see clearly increased uptake in the PSMA+ PC3− PIP tumor relative tothe control (PSMA-negative) PC3-flu tumor at 18.5 h p.i. through 70.5 hp.i. (FIG. 5C through FIG. 5M). Using quantitative real time polymerasechain reaction (qRT-PCR) we measured the relative amounts of PSMA mRNAexpression in extracts of the tumors in mice #1-3, and confirmed thatPC3-PIP tumors (left flank) expressed PSMA mRNA at levels severalmillion times higher than PC3-flu tumors (right flank) (data not shown).Panels 5L and 5M show emission from the intact, living, unshaven animal,while panels 5N and 5O are postmortem studies with organs exposed. Notethat in 5 L one can barely discern the kidneys, a known target site forPSMA (Tasch et al., Crit. Rev. Immunol., vol. 21, pp. 249-261, 2001;Pomper et al., Mol. Imaging, vol. 1, pp. 96-101, 2002; Kinoshita et al.,World J. Surg., vol. 30, pp. 628-636, 2006), while the kidneys areclearly visible in 50 when exposed. A portion of that renal lightemission may be due to clearance of this relatively hydrophiliccompound. The estimated target-to-nontarget ratio (PC-3 PIP vs. PC-3 flulight output) was 10 when comparing the tumors from panel M (70.5 hp.i.).

The experiment in FIG. 6A-FIG. 6T was performed with 10-fold less YC-27administered than in the previous experiment. Despite reducing theconcentration of YC-27, PSMA+ PC3-PIP tumor could be seen clearly at oneday p.i. (FIG. 6A-FIG. 6J, mouse #2, Left Panels). DCIBzL, a known,high-affinity PSMA inhibitor, was co-administered with YC-27 as a testof binding specificity (FIG. 6K-FIG. 6T, mouse #3, Right Panels). Nearlyall of the light emission from target tumor, as well as kidneys, wasblocked, demonstrating the specificity of this compound for PSMA invivo. The estimated target-to-nontarget ratio (PC-3 PIP vs. PC-3 flulight output) was 26 when comparing the tumors from panel F (20.5 hp.i.). By administering 1 nmol to this ˜25 g mouse, we have realized thehigh sensitivity of in vivo optical imaging, rivaling that of theradiopharmaceutical-based techniques. For example, 1 nmol converts to1.6 □g injected. If we synthesized a similar compound labeled with ¹⁸For other radionuclide at 1,000 mCi/□mol (37 GBq/□mol), and administereda standard dose of 200 □Ci (7.4 MBq) to a mouse, we would be injecting0.3 □g.

Interestingly, in mouse #1, which received 10 nmol of YC-27, we observeda small degree of non-specific uptake at the 23 h time point, manifestedas uptake within PSMA-negative PC3-flu tumors. That finding could be dueto enhanced permeability and retention of YC-27. No non-specificuptake/retention was observed at a similar, 20.5 h, time point in mouse#2, which received a 10-fold lower dose. That finding suggests the needfor further optimization of dose and timing for in vivo applications.

Discussion

A wide variety of low molecular weight PSMA-based imaging agents havebeen synthesized, including those using the urea scaffold (Banerjee etal., J. Med. Chem., vol. 51, pp. 4504-4517, 2008; Chen et al., J. Med.Chem., vol. 51, pp. 7933-7943, 2008; Zhou et al., Nat. Rev. DrugDiscov., vol. 4, pp. 1015-1026, 2005; Pomper et al., Mol. Imaging, vol.1, pp. 96-101, 2002; Foss et al., Clin. Cancer Res., vol. 11, pp.4022-4028, 2005; Humblet et al., Mol. Imaging, vol. 4, 448-462, 2005;Misra et al., J. Nucl. Med., vol. 48, pp. 1379-1389, 2007; Mease et al.,Clin. Cancer Res., vol. 14, pp. 3036-3043, 2008; Liu et al., Prostate,vol. 68, pp. 955-964, 2008; Humblet et al., J. Med. Chem., vol. 52, pp.544-550, 2009; Kularatne et al., Mol. Pharm., vol. 6, pp. 790-800, 2009;Hillier et al., Cancer Res., vol. 69, pp. 6932-6940, 2009). Thosecompounds have primarily been radiopharmaceuticals, but optical agentsexist. In two separate studies Humblet et al. reported the synthesis ofmono- and polyvalent NIR fluorescent phosphonate derivatives for imagingPSMA, but little accumulation in PSMA-expressing tumors was evident inthe former study (Humblet et al., Mol. Imaging, vol. 4, pp. 448-462,2005) while no in vivo results were reported in the latter (Humblet etal., J. Med. Chem., vol. 52, pp. 544-550, 2009). Liu et al have alsosynthesized fluorescent phosphonate derivatives and have demonstratedtheir PSMA-binding specificity and intracellular localization in vitro(Liu et al., Prostate, vol. 68, pp. 955-964, 2008). Recently Kularatneet al. have synthesized fluorescent (fluorescein and rhodamine) ureaderivatives that demonstrate PSMA migration to endosomes (Kularatne etal., Mol. Pharm., vol. 6, pp. 790-800, 2009). We arrived at YC-27 basedon structure-activity relationships developed for PSMA-binding ureas,which were focused on improving pharmacokinetics for use in vivo byoptimization of the linker-chelate complex (Banerjee et al., J. Med.Chem., vol. 51, pp. 4504-4517, 2008). Calculated hydrophobicity values(Ghose et al., J. Phys. Chem. A, vol. 102, pp. 3762-3772, 1998) suggestthat YC-27 should be considerably more hydrophobic (A Log D=5.96) thanradiopharmaceuticals such as [¹²⁵I]DCIBzL (A Log D=1.19), perhapsaccounting for its long tumor retention, which is desirable for anoptical imaging agent intended for intraoperative use. We confirmedgreater hydrophobicity of YC-27 relative to DCIBzL through reverse-phaseHPLC (data not shown)

Synthesis of YC-VIII-36

To a solution of YC-VIII-24 (prepared as described in Example 5) (1.5mg, 0.21 □mol) in DMF (1 mL) was added triethylamine (0.005 mL, 35.9μmol), followed by fluorescein isothiocyanate isomer 1 (1 mg, 2.57μmol). After 2 hours at room temperature, the reaction mixture waspurified by HPLC (column, Econosphere C18 5μ, 150×4.6 mm; retentiontime, 15 min; mobile phase, H₂O/CH₃CN/TFA=75/25/0.1; flow rate, 1mL/min) to afford 1.5 mg (72%) of compound YC-VIII-36. ESI-Mass calcdfor C₄₇H₅₇N₆O₁₆S [M+H]⁺ 993.4, found 992.8.

Cell Labeling

PSMA positive PIP cells, and PSMA negative FLU cells were treated withcompound YC-VIII-36 (40 nM) and 4′,6-diamidino-2-phenylindole (DAPI,blue). FIG. 7 shows fluorescence of cells expressing PSMA (greenfluorescence, top left). PIP and FLU cells were treated with bothYC-VIII-36 and PSMA inhibitor PMPA (5 μM), showing inhibition ofcellular fluorescence by PMPA (FIG. 7, bottom).

FIG. 8 shows PC3-PIP cells treated with DAPI (blue) and varyingconcentrations of YC-VIII-36 (green).

FIG. 9 shows time dependent internalization of YC-VIII-36 into PC3-PIPcells treated with YC-VIII-36 (green) and DAPI (blue). The timedependent internalization study was done as described (Liu et al.,Prostate vol. 68, pp. 955-964, 2008) with appropriate modifications.Briefly, PC3-PIP cells were seeded as above. The cells were firstpre-chilled by incubating with ice cold complete growth media and thenincubated with ice cold complete growth media containing 500 nM ofcompound YC-VIII-36 at 40 C for 1 hr. After 1 hr of incubation theexcess compound was removed by washing the wells twice with ice-coldcomplete growth media and then the wells were replenished withpre-warmed complete growth media. The chamber slides containing cellswere incubated for 10 min, 30 min, 60 min and 180 min at 37° C. in ahumidified incubator.

In Vivo Imaging

FIG. 10 shows titration and detection of varying amounts of YC-VIII-36injected subcutaneously into a nude mouse. (IVIS spectrum with 10 secondexposure followed by spectral unmixing).

FIG. 11 and FIG. 12 (top) show fluorescence images of a PSMA+ PC3-PIPand PSMA− PC3-flu tumor-bearing mouse injected intravenously withexemplary compound YC-VIII-36. Compound YC-VIII-36 (150 □g) was injectedinto the tail vein of a nude mouse. The excitation frequency was 465 nmwith a 5 s exposure. Fluorescence emission was measured at 500, 520,540, and 580 nm, followed by spectral unmixing.

FIG. 12 (bottom) show the biodistribution of compound YC-VII-36 (150 □g)180 minutes after injection.

FACS and Cell Sorting

Flow cytometric analysis (FCA): Confluent flasks of PC3-PIP, PC3-flu andLNCap cells were trypsinized, washed with complete growth media (toneutralize trypsin) and counted. Approximately 5 million of each celltype in suspension was incubated with 1 mM of compound YC-VIII-36 for 30min with occasional shaking at 37° C. in the humidified incubator with5% CO₂. After incubation, the cells were washed twice with ice cold KRBbuffer and fixed with 2% paraformaldehyde (ice cold). The samples werestored on ice and protected from light until the FCA was done. FCA wasperformed using a FACS Calibur flow cytometer (Becton Dickinson, SanJose, Calif.). For data acquisition, singlets were gated as theprominent cluster of cells identified from a plot of side scatter (SSC)width versus forward scatter (FSC) width to ensure that cell aggregateswere excluded from analysis. 50,000 total events were counted toestimate the positively stained cells from a plot of Fl-1 (X-axis)versus Fl-2 (Y-axis). All data were analyzed using CellQuest version 3.3software.

Flow sorting: PC3-PIP cells were labeled with 1 mM of compoundYC-VIII-36 for 30 min at 37° C. in the humidified incubator with 5% CO₂.Cells were washed twice with ice cold KRB buffer and stored on ice. Flowsorting was performed using FACS Aria system (Becton Dickinson, SanJose, Calif.) within 10-15 minutes after completion of last wash. Boththe stained (positive) and also the unstained (negative) subpopulationswere collected in sterile tubes containing 3 ml of complete growthmedia. Following sorting, cells were centrifuged, resuspended in warmcomplete growth media, transferred to tissue culture flasks andincubated at 37° C. in the humidified incubator with 5% CO₂ for culture.The sorted subpopulations, “PIP-positive (PIP-pos)” and “PIP-negative(PIP-neg)” cells, were re-analyzed by FCA (as above) at passage 3 forfurther confirmation of their heterogeneity.

Determination of saturation dose inflow cytometry: Approximately 5million cells each of PIP-pos (sorted) and PC3-flu were labeled as abovewith varying doses of compound #. The cells were washed twice with icecold KRB buffer and fixed with 2% paraformaldehyde (ice cold). Thesamples were stored on ice and protected from light till the FCA wasdone. Singlets were gated as above in a plot of SSC vs. FSC to excludethe aggregates. Standard gating was used on X-axis (Fl-1) for analysisof stained cells in all the doses.

PC3-flu, PC3-PIP, and LNCaP cells were treated with compound YC-VIII-36,and analyzed using fluorescence activated cell sorting (FACS) todetermine the percentage of cells expressing PSMA on the cell surface.FIG. 13 shows FACS analysis showing the percent subpopulation of PSMApositive cells in PC3-flu, PC3-PIP, and LNCaP cells. As expected PC3-flu(PSMA−) cells (left) show a very small percentage, while PC3-PIP (PSMA+,center) and LNCaP (PSMA+right) show greater percentages.

PC3-PIP (PSMA+) cells were sorted using FACS following treatment withcompound YC-VIII-36. FIG. 14 shows cell sorting of PC3-PIP cells,including initial percentage (top center), and after 3 passages ofsorting (bottom). Region R² indicates positive PSMA surface expression,as indicated by binding compound YC-VIII-36. The results show anincrease in the percentage of PSMA expressing cells following threerounds of cell sorting.

Determination of detection limit (FIG. 15): PIP-pos cells were mixedwith 10 million of PC3-flu cells in triplicates in different ratios—1 in10⁶, 10⁵, 10⁴, 10 ³ and 10² respectively. All the tubes containing cellsuspensions in complete growth media including controls [10 millionPC3-flu cells with 0% PIP-pos cells and 10 million PIP-pos cells (100%)]were incubated with 100 nM of compound # YC-VIII-36 at 37° C. in thehumidified incubator with 5% CO₂ as above, with occasional stirring. Thecells were washed, fixed with 2% paraformaldehyde as above and analyzedwith LSRII (Becton Dickinson, San Jose, Calif.) for the determination ofdetection limit. Singlets were gated as above in a plot of SSC vs. FSCto exclude the aggregates. 1 million total events were counted toestimate the positively stained cells from plot of Fl-1 (X-axis) versusFl-2 (Y-axis). Two gates, P2 at higher intensity (103 and above) and P3at lower intensity (102-103) on X-axis (Fl-1) was applied for analysisof positive cells. All the data were analyzed using DIVA 6.1.3 software.

Example 4

2-{3-[5-(7-{5-[4-(2-Amino-2-carboxy-ethyl)-[1,2,3]triazol-1-yl]-1-carboxy-pentylcarbamoyl}-heptanoylamino)-1-carboxy-pentyl]-ureido}-pentanedioicacid, compound SRV32. The compound SRV32 was prepared in three stepsfollowing the scheme shown below.

The compound 1 was prepared from literature method (Banerjee et al., JMed Chem, vol. 51, pp. 4504-4517, 2008). To a solution of compound 1(100 mg, 0.107 mmol in 5 ml DMF) was added H-Lys(s-azide)-OH (20 mg,0.107 mmol) (Boc-Lys(Azide)-OH was purchased from Anaspec. The removalof Boc group was done by treating the commercial compound with 1:1TFA:CH₂Cl₂ at room temperature for 4 hr, and the solution was stirredfor 16h at rt. The solvent was removed under vacuum. The solid residuethus obtained was dissolved in 10 ml ethyl acetate and extracted with3×10 mL water. Organic layer was dried under vacuum to get a colorlesssolid as the protected azido urea compound SRV25. ESIMS: 991 [M+1]⁺. Tothe Compound SRV25 (60 mg, 0.06 mmol in 1 ml t-BuOH), was addedN(a)-Boc-L-propargylglycine (Anaspec) (14 mg, 0.012 mmol in 1 mlt-BuOH), followed by Cu(OAc)₂-H₂O (2 mg, 0.012 mmol in 1 ml water) andsodium ascorbate (4.75 mg, 0.024 mmol in 1 ml water) and the mixturestirred at room temperature for 12 h. The product was extracted intoCH₂Cl₂ and washed twice with aqueous NaCl. The aqueous phases werere-extracted with CH₂Cl₂. The organic phases were combined, dried overNa₂SO₄ and evaporated. The product, compound SRV29, was purified by asilica gel pipette column eluted with solution of 90/10 CH₂Cl₂/MeOH.ESIMS: Calcd for C₆₀H₈₂N₈O₁₈ 1203.57, found 1204[M+1]⁺.

The compound SRV29 was dissolved in 2 ml 1/1 CHCl₃/TFA and stirredovernight. The solution was removed under vacuum to get a colorlesssolid. The solid was washed 3 times with 5 ml CH₂Cl₂ to removeimpurities. The crude solid, compound SRV32 was further purified by HPLCusing an 85/15 water/acetonitrile (0.1% TFA in each) flow rate 4 ml,R=10.2 min. ESMS: 742.77 [M+1]⁺, ¹H NMR (D₂O) δ: 7.46 (M, 1H), 5.2 (m,2H) 4.35 (m, 1H), 4.26 (m, 1H), 4.18 (m, 1H), 3.80-3.70 (m, 1H), 3.18(t, J=6 Hz, 2H), 2.69 (m, 2H), 2.51 (t, J=7.6 Hz, 2H), 2.40-2.18 (m,25H).

Radiolabeling with Tc-99m was performed by the same procedure describedpreviously (Banerjee et al, J Med Chem, vol. 51, pp. 4504-4517, 2008).

Biodistribution and Imaging

A single SCID mouse implanted with both a PC-3 PIP (PSMA+) and a PC-3flu (PSMA−) xenograft was injected intravenously with compound^(99m)Tc-SRV32 in saline. At 0.5 hr, 1 hr, 2 h, and 5 h p.i. the mousewas anesthetized with isoflurane and maintained under 1% isoflurane inoxygen. The mouse was positioned on the X-SPECT (Gamma Medica,Northridge, Calif.) gantry and was scanned using two low energy,high-resolution pinhole collimators (Gamma Medica) rotating through 360°in 6° increments for 45 seconds per increment. All gamma images werereconstructed using Lunagem software (Gamma Medica, Northridge, Calif.).

Immediately following SPECT acquisition, the mice were then scanned byCT (X-SPECT) over a 4.6 cm field-of-view using a 600 μA, 50 kV beam. TheSPECT and CT data were then coregistered using the supplier's software(Gamma Medica, Northridge, Calif.) and displayed using AMIDE(http://amide.sourceforge.net/). Data were reconstructed using theOrdered Subsets-Expectation Maximization (OS-EM) algorithm.

Tissue biodistribution was measured. Results are summarized in thefollowing table. ^(99m)Tc-SRV32 exhibited high uptake (˜7% ID/g at 30minutes), and good clearance from non-target tissues.

Tissue 30 min 60 min 120 min 300 min blood 1.38 ± 0.4 0.63 ± 0.1 0.61 ±0.3 0.19 ± 0.1 liver 14.26 ± 1.0  9.81 ± 2.0 5.65 ± 0.5 3.06 ± 0.6stomach 0.77 ± 0.1  0.42 ± 0.09 0.29 ± 0.1 0.18 ± 0.1 spleen 26.10 ±9.0  17.31 ± 6.6  5.80 ± 1.9 1.26 ± 0.5 kidney 139.53 ± 17.2  144.65 ±15.1  151.23 ± 37.1  80.00 ± 8.4  muscle 0.56 ± 0.1 0.40 ± 0.2 0.16 ±0.1 0.51 ± 0.6 small 1.94 ± 1.1 0.74 ± 0.3 0.39 ± 0.2 0.26 ± 0.2intestine large 0.61 ± 0.1 0.36 ± 0.1 0.53 ± 0.4 2.96 ± 1.3 intestinebladder 1.07 ± 0.1 3.09 ± 2.6 5.39 ± 7.1 2.74 ± 2.1 PC-3 PIP 6.67 ± 1.65.32 ± 1.2 3.77 ± 0.8 2.19 ± 0.5 PC-3 flu 0.75 ± 0.2 0.45 ± 0.3 0.35 ±0.2 0.43 ± 0.4

A single SCID mouse implanted with a PSMA+LnCaP xenograft was injectedintravenously with compound ^(99m)Tc-SRV32 in saline. At 0.5 hr. and 3.5hr p.i. the mouse was anesthetized with isoflurane and maintained under1% isoflurane in oxygen. The mouse was positioned on the X-SPECT (GammaMedica, Northridge, Calif.) gantry and was scanned using two low energy.high-resolution pinhole collimators (Gamma Medica) rotating through 360°in 6° increments for 45 seconds per increment. All gamma images werereconstructed using Lunagem software (Gamma Medica, Northridge, Calif.).Immediately following SPECT acquisition, the mice were scanned by CT(X-SPECT) over a 4.6 cm field-of-view using a 600 μA, 50 kV beam. TheSPECT and CT data were then coregistered using the suppliers software(Gamma Medica. Northridge, Calif.) and displayed using AMIDE(http://amide.sourceforge.net/). Data were reconstructed using theOrdered Subsets-Expectation Maximization (OS-EM) algorithm. Images areshown in FIG. 16.

Comparative Example 1

Under the same conditions, tumor uptake for compound ^(99m)Tc-L1, shownbelow, was determined. Results are summarized in the following table.The data show that while ^(99m)TC-L1 shows good retention, compound^(99m)Tc-SRV32 has greater retention in vivo both for target tumor andnontarget tissues, and lower GI uptake than the previous ^(99m)TcL1compound at initial time points.

Tissue 30 min 60 min 120 min 300 min PC-3 PIP 7.9 ± 4   3.9 ± 0.6 2.0 ±0.8 0.8 ± 0.5 PC-3 flu 0.3 ± 0.2 0.2 ± 0.1 0.05 ± 0.02 0.01 ± 0.01

Example 5 ⁶⁸Ga Compounds General

Solvents and chemicals obtained from commercial sources were ofanalytical grade or better and used without further purification. Allexperiments were performed in duplicate or triplicate to ensurereproducibility. Analytical thin-layer chromatography (TLC) wasperformed using Aldrich aluminum-backed 0.2 mm silica gel Z19, 329-1plates and visualized by ultraviolet light (254 nm), 12 and 1% ninhydrinin EtOH. Flash chromatography was performed using silica gel purchasedfrom Bodman (Aston Pa.), MP SiliTech 32-63 D 60 Å. ¹H NMR spectra wererecorded on either a Varian Mercury 400 MHz or on a Bruker Ultrashield™400 MHz spectrometer. Chemical shifts (6) are reported in ppm downfieldby reference to proton resonances resulting from incomplete deuterationof the NMR solvent. Low resolution ESI mass spectra were obtained on aBruker Daltonics Esquire 3000 Plus spectrometer. Higher-resolution FABmass spectra were obtained on a JOEL JMS-AX505HA mass spectrometer inthe mass spectrometer facility at the University of Notre Dame. Opticalrotation was measured on a Jasco P-1010 polarimeter. Infrared spectrawere obtained on a Bruker Tensor 27 spectrometer. High-performanceliquid chromatography (HPLC) purification of new compounds was performedusing a Phenomenex C₁₈ Luna 10-×250 mm² column on a Waters 600E Delta LCsystem with a Waters 486 tunable absorbance UV/Vis detector, bothcontrolled by Empower software.

For purification of radiolabeled [⁶¹Ga]SRV100, a Varian Microsorb-Mv C₁₈250×4.6 mm² column was used. HPLC was performed using the followingisocratic conditions: For Method 1, the mobile phase was 80% solvent A(0.1% TFA in water) and 20% solvent B (0.1% TFA in CH₃CN), flow rate 4mL/min; for Method 2, the mobile phase was 80% solvent A and 20% solventB, flow rate 1 mL/min. Method 1 was used for purification of compoundsSRV27, [^(69/71)Ga]SRV27, SRV100, [^(69/71)Ga]SRV100 and [⁶⁸Ga]SRV27.

For purification of [⁶⁸Ga]SRV100 Method 2 was used. For radiosyntheticpurification, HPLC was performed on a Varian Prostar System (Palo Alto,Calif.), equipped with a model 490 UV absorbance detector and a BioscanNaI scintillation detector connected to a Bioscan Flow-count systemcontrolled by Empower software.

Radiochemistry

⁶⁸Ga labeling protocol for compound SRV27 was done following aliterature procedure (Zhemosekov et al., J Nucl Med, vol. 48, pp.1741-1748, 2007). A detailed description is given below.

1. 13.2 mCi of ⁶⁸Ga in 7 mL of 0.1N HCl were obtained from more than1-year-old 740-MBq generator. The solution was transferred on acation-exchange cartridge, Phenomenex Strata-X-C tubes (33 μm strongcation exchange resin, part no. 8B-S029-TAK, 30 mg/l ml).2. The column was eluted with 5 ml of a solution of 20/80 0. IONhydrochloric acid/acetone. The eluant remaining on the cation-exchangerwas removed by passage of nitrogen. These two processes aimed to removemost of the remaining chemical and radiochemical impurities from theresin, whereas Ga(III) should quantitatively remain on the column.3. The column was filled with 150 μL of a 2.4/97.6 0.05N HCl/acetonesolution. About 2 min standing appeared to be best for completedesorption of the Ga(III) from the resin into the liquid phase. Anadditional 250 μL of this mixture were applied, and the purified⁶⁸Ga(III) was obtained in 400 μL of this eluent overall.4. The fraction (400 μL eluent) was used directly for the labeling ofDOTA-urea compound. The processed activity was added to 500 μL pure H₂Oin a standard glass reagent vial containing 100 μl (92 nmol, 1 mg/mLsolution) of ligand. No buffer solution was added. The reaction vial washeated at 95° C. for 10 min. The complexation was monitored by injectingaliquots of 100 μl (210 ρCi) of the solution in HPLC. Productobtained=160 μCi. Radiochemical Yield=(160/210)×100=76.19% (withoutdecay correction). Solvent system 80/20 water/acetonitrile (0.1% TFA ineach). R_(t) (retention time)=25 min for the compound and R_(t)=19 minfor the free ligand. Product obtained=5.92 MBq. For [⁶⁸Ga]SRV27,radiochemical yield: 76.2% (without decay correction). HPLC wasperformed by Method 1 as described in the General experimental section.R_(t)=25 min for the desired product and R_(t)=19 min for the freeligand. For [⁶⁸Ga]SRV100, radiochemical yield: 70%. HPLC was performedby Method 2 as mentioned in General experimental section. R_(t)=22.5 minfor the desired product and R_(t)=16 min for the free ligand.

Cell Lines and Tumor Models

PC-3 PIP (PSMA+) and PC-3 flu (PSMA) cell lines were obtained from Dr.Warren Heston (Cleveland Clinic) and were maintained as previouslydescribed (Mease et al., Clin Cancer Res, vol. 14, pp. 3036-3043, 2008).LNCaP cells were obtained from American Type Culture Collection (ATCC,Manassas, Va.) and were maintained as per ATCC guidelines. All cellswere grown to 80-90% confluence before trypsinization and formulation inHank's Balanced Salt Solution (HBSS, Sigma, St. Louis, Mo.) forimplantation into mice.

Animal studies were undertaken in compliance with institutionalguidelines related to the conduct of animal experiments. Forbiodistribution studies of [Ga]SR V27, and [⁶⁸Ga]SRV100 and imagingstudies of [⁶⁸Ga]SRV100, male SCID mice (NCI) were implantedsubcutaneously with 1-5×10⁶ PSMA+ PC-3 PIP and PSMA− PC-3 flu cellsbehind either shoulder. For imaging studies of [⁶⁸Ga] SRV27, male SCIDmice (NCI) were implanted subcutaneously with 5×10⁶ LNCaP cells behindthe right shoulder. Mice were imaged or used in biodistribution studieswhen the tumor xenografts reached 3-5 mm in diameter.

Synthesis of SRV27

[2-{3-[5-(7-{1-Benzyloxycarbonyl-5-[2-(4,7,10-tris-carboxymethyl1,4,7,10tetraazacyclododec-1-yl)-acetylamino]-pentylcarbamoyl}-heptanoylamino)-1-carboxy-pentyl]-ureido}-pentanedioicacid (SR V27). Compound SRV27 was prepared in three steps according tothe following scheme.

Compound 1 was prepared according to a literature method (Banerjee et JMed Chem, vol. 51, pp. 4504-4517, 2008). To a solution of compound 1(100 mg, 0.11 mmol in 5 mL DMF) was added H-Lys(Boc)-OBz (36 mg, 0.11mmol) (Hamachi et al., Chem. Eur. J., vol. 5, pp. 1503-1511, 1999). Thesolution was stirred for 16 h at ambient temperature. The solvent wasremoved under vacuum. The solid residue thus obtained was dissolved in10 mL ethyl acetate and extracted with 3×10 mL water. The organic layerwas dried under vacuum to provide a colorless solid ESIMS: 1154 [M+1]⁺.This crude compound was dissolved in 3 mL CHCl₃ followed by addition of3 mL TFA at 0° C. The solution was allowed to stir overnight at ambienttemperature. The volume of the solution was reduced under vacuum and thesolid residue was washed with 3×5 mL CH₂Cl₂ to remove impurities. Thecolorless solid residue, 3, was dried under vacuum to give 80 mg ofcompound 3. Compound 3 was purified further by using a 2 g Sep Pak C₁₈cartridge with a solution of 85/15 water/acetonitrile (0.1% TFA ineach). ¹H NMR (D₂O, δ): 7.5 (bm, 5H), 4.27 (m, 1H), 4.12 (m, 1H), 3.99(m, 1H), 3.04 (m, 4H), 2.38 (m, 2H), 2.3-1.0 (m, 27H). ESIMS: 694[M+1]⁺. To a solution of DOTA-mono-NHS (54 mg, 0.11 mmol in 5 mL DMF)was added 3 (80 mg, 0.08 mmol) and TEA (60 μL, 0.43 mmol) and thesolution was allowed to stir for 16 h at ambient temperature. Solventwas removed under vacuum and the crude solid, SRV27, was purified byHPLC Method 1, retention time 19 min.

Yield: μ400%. ESMS: 1080[M+1]⁺, HRESI⁺-MS: Calcd. for C₄₉H₇₇N₉O₁₈,1080.5487 [M+H], found: 1080.5459. ¹H NMR (D₂O) δ: 7.88 (m, 4H),4.26-4.1 (m, 5H), 3.45-3.18 (m, 16H), 2.52-2.43 (m, 16H), 2.40-2.18 (m,25H). 13C (CD₃CO₂D) d: 177.5, 177.6, 175.3, 172.3, 160.6, 160.2, 159.8,159.5, 135.5, 128.5, 128.4, 119.9, 117, 114.0, 111.3, 67.3, 55.5, 53.1,51.0, 49.9, 30.7, 28.0, 26.4, 25.1.

2-{3-[5-(7-{1-Benzyloxycarbonyl-5-[2-(4,7,10-tris-carboxymethyl-1,4,7,10tetraaza-cyclododec-1-yl)-acetylamino]-pentylcarbamoyl}-heptanoylamino)-1-carboxy-pentyl]-ureido}-pentanedioicacid Gallium (HI), SRV31 ([^(69/71)Ga]-SRV27). To a solution of GaNO₃(10 mg, 39 μmol) in deionized water was added compound SRV27 (4.2 mg, 39μmol) in 1 mL deionized water and the resulting solution was heated inboiling water for 10 min. The solvent was evaporated to dryness and thecrude residue was purified by HPLC using an 80/20 water/acetonitrile(0.1% TFA in each), flow rate 8 ml/min. Retention time for the productwas at 12 min. Yield: ˜35% ESMS: 1146[M+1]⁺, ¹H NMR (D₂O) δ: 7.88 (m,4H), 4.26-4.1 (m, 5H), 3.45 (m, 8H) 3.18 (m, 8H), 2.69 (m, 8H), 2.51 (m,8H), 2.40-2.18 (m, 25H).

SRV100

2-[3-(1-Carboxy-5-{7-[5-carboxy-5-(3-phenyl-2-{3-phenyl-2-[2-(4,7,1I0-tris-carboxymethyl-1,4,7,10-tetraaza-cyclododec-1-yl)-acetylamino]-propionylamino}-propionylamino)-pentylcarbamoyl]-heptanoylamino}-pentyl)-ureido]-pentanedioicacid, (SRV100). Compound SRV100 was prepared according to the schemeshown in FIG. 19. Fmoc-Lys(Boc)-Wang resin (100 mg, 0.43 mM) was allowedto swell with CH₂Cl₂ (3 mL) followed by DMF (3 mL). A solution of 20%piperidine in DMF (3×3 mL) was added to the resin that was then shakengently on a mechanical shaker for 30 min at ambient temperature. Theresin was washed with DMF (3×3 mL) and CH₂Cl₂ (3×3 mL). Formation offree amine was assessed by the Kaiser test (Kaiser et al., Anal Biochem,vol. 34, pp. 595-598, 1970). After swelling the resin in DMF, a solutionof Fmoc-Phe-OH (3 eq), HBTU (3 eq), HOBt (3 eq), and DIPEA (4.0 eq) inDMF was added and gently shaken for 2 h. The resin was then washed withDMF (3×3 mL) and CH₂Cl₂ (3×3 mL). The coupling efficiency was assessedby the Kaiser Test. That aforementioned sequence was repeated for twomore coupling steps with Fmoc-Phe-OH and DOTA-(t-butyl ester)₃-CO₂H. Theresulting compound was cleaved from the resin using TFA: CH₂Cl₂ (1:1)and concentrated under vacuum to produce the free amine. Theconcentrated product was purified by using a C₁₈ SepPak Vac 2 g column.The product was eluted with a solution 70/30 water/acetonitrile (0.1%TFA in each). ESIMS: 827 [M+1]⁺. Lyophilized amine (10 mg, 12 μmol in 2mL DMF) was added to 1 (prepared separately) (20 mg, 21.4 μmol in 1 mLDMF) followed by TEA (214 μmol, 30 μL) and then stirred at 25° C. for 16h. After solvent removal, solid residue was treated with 3 mL TFA:CH₂Cl₂to remove the PMB group. The residue was washed 2×5 mL CH₂Cl₂ to removeimpurities. The colorless solid residue thus obtained was purified by aC₁₈ SepPak Vac 2 g column using an eluent of 70/30 water/acetonitrile(0.1% TFA in each) to produce SRV100 (SR-V-100). The product was furtherpurified using preparative RP-HPLC by Method 1, retention time 17 min.Yield: ˜ 30%. ESMS m/Z: 1284[M+H], HRESI+-MS: Calcd. for C₆₈H₉₀N₁₁O₂₀,1284.6365 [M+H], found: 1284.6358. ¹H NMR (CD₃CO₂D) δ: 7.35-7.20 (m,10H), 4.86 (bm, 2H), 4.57-4.46 (4H), 4.4-2.8 (m, 14H), 2.51 (t, 2h),2.4-1.2 (m, 28H). 13C (CD₃CO₂D) δ: 176.5, 177, 177.06, 177.6, 173.6,173.24, 161.3, 160.92, 160.53, 160.14, 159.77, 137.95, 137.06, 130.5,129.5, 127.9, 127.71, 120.8, 118.0, 115.1, 112.3, 56.1, 55.5, 53.5,53.3, 40.1, 38.8, 36.832.6, 31.8, 30.7, 29.42, 27.9, 26.53.

2-[3-(1-Carboxy-5-{7-[5-carboxy-5-(3-phenyl-2-{3-phenyl-2-[2-(4,7,10-tris-carboxymethyl-1,4,7,10tetraaza-cyclododec-1-yl)-acetylamino]-propionylamino}-propionylamino)-pentylcarbamoyl]-heptanoylamino}-pentyl)-ureido]-pentanedioicacid Gallium (III), [^(69/71)Ga]SRV100. This compound was preparedaccording to the same general procedure as described for[^(69/71)Ga]SRV27. Compound [⁶⁹⁷¹Ga]SRV100 was purified by Method 1,retention time 22 min. Yield: ˜30%. ESMS m/Z: 1351[MH-H]⁺, HRESI⁺-MS:Calcd. For C₆₈H₈₆GaNnNaO₂₀, 1372.5204 [M+Na]⁺, found: 1372.5199.

Compound Characterization—Lipophilicity

Partition coefficients, Log_(0/W) (pH=7.4) values were determinedaccording to a literature procedure (Antunes et al., Bioconjug Chem,vol. 18, pp. 84-92, 2007). Briefly, a solution of either [⁶⁸Ga]SRV27 or[⁶⁸Ga]SRV100 was added to a presaturated solution of 1-octanol (5 mL)mixed with phosphate buffered saline (PBS) (5 mL) in a 15 mL centrifugetube.

After vigorously shaking the mixture, it was centrifuged at 3,000 rpmfor 5 min. Aliquots (100 μL) were removed from the two phases and theradioactivity was measured in a γ-counter, 1282 Compugamma CS (LKB,Wallac, Turku. Finland).

On analysis of the reaction mixture by HPLC, the retention time of theradiolabeled compound was slightly longer than the corresponding freeligand. The specific radioactivity of purified [⁶⁸Ga]SRV27 and[⁶⁸Ga]SRV100 was between 3.0 and 6.0 MBq/nmol.

The log P_(octanol/water) values for [⁶⁸Ga]SRV27 and [⁶⁸Ga]SRV100 wereapproximately −3.9 as determined by the shake-flask method (Antunes etal., Bioconjug Chem, vol. 18, pp. 84-92, 2007). However, using an HPLCmethod, we found that the HPLC retention times for SRV100 (28 min) and[^(69/71)Ga]SRV100 (32 min) were longer than for SRV27 (19 min) and[^(69/71)Ga]SRV27 (24 min). It is evident that SRV100 and thecorresponding gallium compound were more lipophilic than SRV27 and itsgallium-labeled analog, which is reasonable in light of the presence oftwo phenylalanine residues in the long linker of SRV100, while SRV27 hasonly one lysine residue protected as the benzyl ester.

Cell Binding Assay

Ki values for SRV27, [^(69, 71) Ga]SRV27, SRV100 and [^(69, 71)Ga]SRV100were determined using a competitive N-acetyl aspartyl glutamate (NAAG)fluorescence cell binding assay adapted from the literature (Kozikowskiet al., J Med Chem, vol. 47, pp. 1729-1738, 2004). All compounds werefound to be strong inhibitors of PSMA. Compounds SRV27 and[^(69, 71)Ga]SRV27 had inhibitory capacities of 2.9 nM and 29 nM,respectively. For SRV100 and [^(69, 71)Ga]SRV100, values were 1.23 nMand 0.44 nM, respectively.

Ex Vivo Biodistribution

PSMA+ PC-3 PIP and PSMA− PC-3 flu xenograft-bearing SCID mice wereinjected via the tail vein with 30 μCi (1.1 MBq) of [⁶⁸Ga]SRV27 or[⁶⁸Ga]SRV100. In case each four mice were sacrificed by cervicaldislocation at 30, 60, 120, 180 min p.i. For [⁶⁸Ga]SR V27 and at 5, 60,120, 180 min p.i. for [⁶⁸Ga]SRV100. The heart, lungs, liver, stomach,pancreas, spleen, fat, kidney, muscle, small and large intestines,urinary bladder, and PC-3 PIP and flu tumors were quickly removed. A 0.1mL sample of blood was also collected. Each organ was weighed, and thetissue radioactivity was measured with an automated gamma counter (1282Compugamma CS, Pharmacia/LKB Nuclear, Inc., Gaithersburg, Md.). The %ID/g was calculated by comparison with samples of a standard dilution ofthe initial dose. All measurements were corrected for decay.

Compound [⁶⁸Ga]SRV27 was assessed for its pharmacokinetics ex vivo insevere-combined immunodeficient (SCID) mice bearing both PSMA+ PC3-PIPand PSMA−PC3− flu xenografts (Chang et al., Cancer Res, vol. 59, pp.3192-3198, 1999). Table 1 shows the percent injected dose per gram (%ID/g) of radiotracer in selected organs for [⁶⁸Ga]SR V27.

TABLE 1 Ex vivo tissue biodistribution of [⁶⁸Ga]SRV27 Tissue 30 min 60min 120 min 180 min blood 2.20 ± 0.90 1.93 ± 0.70 0.80 ± 0.30 0.62 ±0.34 heart 0.70 ± 0.13 0.50 ± 0.08 0.21 ± 0.08 0.20 ± 0.02 liver 0.84 ±0.24 0.83 ± 0.10 0.42 ± 0.07 0.50 ± 0.03 stomach 0.73 ± 0.13 0.75 ± 0.320.24 ± 0.07 0.24 ± 0.05 spleen 4.90 ± 1.10 3.35 ± 1.20 0.43 ± 0.19 0.32± 0.13 kidney 97.19 ± 16.07 64.68 ± 4.10  5.35 ± 2.12 2.13 ± 0.11 muscle0.46 ± 0.16 0.25 ± 0.07 0.08 ± 0.04 0.05 ± 0.01 small intestine 0.79 ±0.12 0.70 ± 0.34 0.26 ± 0.11 0.34 ± 0.20 large intestine 0.77 ± 0.140.95 ± 0.53 0.34 ± 0.10 0.46 ± 0.10 bladder 8.96 ± 5.30 25.29 ± 8.63 2.70 ± 4.02 5.39 ± 2.98 PC-3 PIP 3.78 ± 0.90 3.32 ± 0.33 1.31 ± 0.061.10 ± 0.19 PC-3 flu flu 0.82 ± 0.20 0.67 ± 0.08 0.41 ± 0.09 0.39 ± 0.02PIP:flu 4.61 4.93 3.24 2.77 PIP:muscle 8.30 13.13 17.40 20.37 flu:muscle1.80 2.67 5.37 7.34

Compound [⁶⁸Ga]SRV27 showed clear PSMA-dependent binding in PSMA+PC3 PIPxenografts, reaching a maximum uptake of 3.78±0.90 (SEM) % ID/g at 30min post-injection (p.i.). The blood, spleen and kidney displayedhighest uptake at 30 min. By 60 min, the urinary bladder showed highestuptake, however, this uptake represents excretion at all time points.The high values noted in kidney are partially due to high expression ofPSMA within proximal renal tubules (Silver et al., Clin Cancer Res, vol.3, pp. 81-85, 197: Slusher et al., J Comp Neurol, vol. 315, pp. 271-229,1992). Rapid clearance from the kidneys was demonstrated, decreasingfrom 97.19±16.07% ID/g at 30 min to 2.31±0.11% ID/g at 3 h. Theradioactivity in the PSMA+PIP tumor cleared more slowly, from itsaforementioned value at 30 min to 1.08±0.19% ID/g at 3 h.

Compound [⁶⁸Ga]SRV100 was also investigated for its pharmacokineticcharacteristics in tumor bearing mice at 5 min, 1 h, 2 h and 3 h p.i.Table 2 shows the % ID/g of radiotracer in selected organs for[⁶⁸Ga]SRV100.

TABLE 2 Ex vivo tissue biodistribution of [⁶⁸Ga]SRV100 Tissue 5 min 60min 120 min 180 min blood 6.28 ± 0.08 0 .41 ± 0.05 0.15 ± 0.07 0.13 ±0.01 heart 2.01 ± 0.24 0.19 ± 0.07 0.05 ± 0.03 0.03 ± 0.01 lung 4.59 ±0.68 0.74 ± 0.54 0.20 ± 0.05 0.14 ± 0.03 liver 1.57 ± 0.16 0.24 ± 0.090.19 ± 0.03 0.14 ± 0.02 stomach 2.38 ± 0.35 0.38 ± 0.16 0.18 ± 0.02 0.04± 0.02 pancreas 1.52 ± 0.19 0.25 ± 0.14 0.08 ± 0.03 0.04 ± 0.02 spleen5.17 ± 2.22 2.43 ± 1.07 0.78 ± 0.15 0.34 ± 0.09 fat 1.03 ± 0.02 0.40 ±0.04 0.08 ± 0.02 0.02 ± 0.01 kidney 64.75 ± 12.00 26.57 ± 10.93 12.25 ±1.79  10.04 ± 1.22  muscle 1.58 ± 0.33 0.15 ± 0.08 0.03 ± 0.02 0.00 ±0.01 small intestine 2.04 ± 0.25 0.23 ± 0.05 0.09 ± 0.04 0.06 ± 0.03large intestine 2.02 ± 0.49 0.50 ± 0.70 0.12 ± 0.03 0.12 ± 0.03 bladder5.97 ± 1.50 7.65 ± 3.34 1.41 ± 1.17 0.75 ± 0.54 PC-3 PIP 6.61 ± 0.552.80 ± 1.32 3.29 ± 0.77 1.80 ± 0.16 PC-3 flu 2.63 ± 0.51 0.16 ± 0.080.18 ± 0.03 0.12 ± 0.03 PIP:flu 2.50 17.30 18.28 15.20 PIP:muscle 4.1723.27 122.13 436.29 flu:muscle 1.67 1.34 6.68 28.70

As for [⁶⁸Ga]SRV27. [⁶⁸Ga]SRV100 showed PSMA-dependent tumor uptake.After a peak, flow-related, uptake at 5 min p.i. of 6.61±0.55%,[⁶⁸Ga]SRV100 demonstrated a 2 h tumor uptake value of 3.29±0.77%, whichdropped to 1.80±0.16% at 3 h. Uptake in blood was high at 5 min andrapidly washed out within 1 h. Non-target organs such as kidney, spleenand lung showed high uptake at 5 min and rapidly washed out with time.With the exception of the kidneys and spleen, clearance from blood andnormal organs was faster for [⁶⁸Ga]SRV100 than for [⁶⁸Ga]SRV27. Again,high kidney uptake is associated with high expression of PSMA withinproximal renal tubules (Silver et al., Clin Cancer Res, vol. 3, pp.81-85, 197; Slusher et al, J Comp Neurol, vol. 315, pp. 271-229, 1992).Similar to [⁶⁸Ga]SRV27, [⁶⁸Ga]SRV100 demonstrated faster clearance ofradioactivity from kidney than from the PSMA+tumor. However, the rate ofclearance from kidney for [⁶⁸Ga]SRV100 was much slower than for[⁶⁸Ga]SRV27, i.e., 65±12% at 5 min p.i. and 10.04±1.22% at 3 h.

Small Animal PET Imaging

A single SCID mouse implanted with a PSMA+LNCaP xenograft was injectedintravenously with 0.2 mCi (7.4 MBq) of [6¹Ga]SRV27 in 200 μL 0.9% NaCl.At 0.5 h p.i., the mouse was anesthetized with 3% isoflurane in oxygenfor induction and maintained under 1.5% isoflurane in oxygen at a flowrate of 0.8 L/min. The mouse was positioned in the prone position on thegantry of a GE eXplore VISTA small animal PET scanner (GE Healthcare,Milwaukee, Wis.). Image acquisition was performed using the followingprotocol: The images were acquired as a pseudodynamic scan, i.e., asequence of successive whole-body images were acquired in three bedpositions for a total of 120 min. The dwell time at each position was 5min, such that a given bed position (or mouse organ) was revisited every15 min. An energy window of 250-700 keV was used. Images werereconstructed using the FORE/2D-OSEM method (two iterations, 16 subsets)and included correction for radioactive decay, scanner dead time, andscattered radiation. After PET imaging, the mobile mouse holder wasplaced on the gantry of an X-SPECT (Gamma Medica Ideas, Northridge,Calif.) small animal imaging device to acquire the corresponding CT.Animals were scanned over a 4.6 cm field-of-view using a 600 μA, 50 kVbeam. The PET and CT data were then co-registered using Amira 5.2.0software (Visage Imaging Inc., Carlsbad, Calif.).

Imaging studies of [⁶⁸Ga]SRV100 and blocking studies of [⁶⁸Ga]SRV27 werecarried out on PSMA+ PC-3 PIP and PSMA− PC-3 flu xenograft-bearing SCIDmice or PSMA+ PC-3 PIP (25.9 MBq in 100 μL NaCl) xenograft-bearing SCIDmice. At 30 min, 1 h and 2 h p.i. the mice were anesthetized andwhole-body images were obtained using the PET scanner as mentionedabove, in two bed positions, 15 min at each position for a total of 30min using the same energy window. Images were reconstructed andco-registered with the corresponding CT images using the same methods asdescribed above.

FIGS. 17 and 18 demonstrate the high target selectivity of [⁶⁸Ga] SRV27and [⁶⁸Ga]SRV100 by delineating the PSMA+tumors. Although a PSMA−control tumor was not included in FIG. 17, a separate blocking study wasperformed for [Ga]SRV27, in which an animal pre-treated with 50 mg/kg ofthe known PSMA− binding ligand, 2-(phosphonomethyl)pentanedioic acid(2-PMPA) (Jackson et al., J Med Chem. vol. 39, pp. 619-622, 1996), didnot demonstrate PSMA+tumor uptake, attesting to the binding specificityof this compound. The more quantitative, ex vivo studies of [⁶⁸Ga]SRV27and [⁶⁸Ga]SRV100 further supported high PSMA target specificity,demonstrating target-to-nontarget (PIP/flu) ratios of approximately 5and 18 at 1 h and 2 h p.i., respectively. One hour and 2 h PSMA+tumoruptake values for these compounds, 3.32±0.33% and 3.29±0.77%,respectively, for [⁶⁸Ga]SRV27 and [Ga]SRV100, are comparable to otherradiometallated PSMA inhibitors (Banerjee et al., J Med Chem, vol. 51,pp. 4504-4517, 2008). As shown in FIGS. 17 and 18 those values aresufficient for clear tumor imaging. Notably, PIP tumors contain aboutone order of magnitude lower PSMA than LNCaP tumors (data not shown),which are often employed to assess for binding specificity ofPSMA-targeting agents. PIP/flu is the preferred comparison as both arederived from PC-3 cells, providing a more controlled study.

Intense radiotracer uptake was seen only in the kidneys and tumor forboth [⁶⁸Ga]SRV27 (FIG. 17) and [⁶⁸Ga]SRV100 (FIG. 18). As noted abovefor the ex vivo study, the intense renal uptake was partially due tospecific binding of the radiotracer to proximal renal tubules (Silver etal., CHn Cancer Res, vol. 3. pp. 81-85, 197: Slusher et al., J CompNeurol, vol. 315, pp. 271-229, 1992) as well as to excretion of thishydrophilic compound. Apart from the kidneys, only the PSMA+tumordemonstrated significant radiotracer uptake.

Discussion

Because of its demonstrated clinical utility and the appearance of dualmodality (PET/computed tomography (CT)) systems, clinical PET imaginghas been accelerating worldwide and may soon become the dominanttechnique in nuclear medicine. PET isotopes tend to be short-lived andenable synthesis of “physiologic” radiotracers, namely, those thatincorporate ¹⁵0, ¹³N or ¹¹C, enabling precise conformity to the tracerprinciple. Being essentially isosteric to H, F enables nearlytracer-level studies, with important caveats, particularly for[¹⁸F]fluorodeoxyglucose (FDG), which is by far the most commonly usedradiopharmaceutical for PET. But, in part because FDG does notaccumulate well within many tumor types, including prostate cancer,there has been a re-emergence in the development of radiometallatedpeptides, often employing ^(99m)Tc, that target G-protein coupledreceptors. Gallium-68 provides a link between PET and single photonemission computed tomography (SPECT) since metal chelating methodologyneeded for ^(99m)Tc can also be applied to ⁶⁸Ga. A further analogy isthe convenience of use of a ⁶⁸Ge/⁶⁸Ga generator (PET), as with⁹⁹Mo^(99m)Tc (SPECT), to provide readily available isotope. with no needfor an in-house cyclotron. Although ¹⁸F-labeled, low molecular weightPSMA inhibitors have shown promise in preclinical imaging studies (Measeet al., Clin Cancer Res, vol. 14, pp. 3036-3043, 2008; Lapi et al., JNucl Med, vol. 50, pp. 2042-2048, 2009), the availability ofgenerator-produced ⁶⁸Ga and the extension to PET from our published^(99m)Tc-labeled series of PSMA-binding radiometallated imaging agents(Banerjee et al., J Med Chem, vol. 51, pp. 4504-4517, 2008) provided therationale for this study.

Example 6

Compound SRV27 and SRV100 were prepared as described in Example 5.In-111 labeling was generally performed by treatment of SRV27 or SRV100or SRV73 with ¹¹¹InCl₃, in 200 mM aqueous NaOAc −60° C. for 30 minutes.Specifically, for SRV27, 60 μl of SRV27 (2 mg/mL, sodium acetate) wascombined with 100 μl sodium acetate and 3 mCi ¹¹¹InCl₃ in a 1.5 mlEppendorf tube and left at for 60° C. for 30 min. The radiolabeledproduct was diluted with 800 μl water and purified by HPLC.Radiolabeling yield is 1.7 mCi (−57%) and radiochemical purity was>99.9%

SRV73

Compound SRV73 was prepared by the method outlined in the scheme below.Compound SRV73 is a bimodal compound having a fluorescent dye moiety anda metal chelating moiety

Small Animal PET Imaging

SPECT imaging experiments for [¹¹¹In]SRV27, [¹¹¹In]SRV100 and[¹¹¹In]SRVTS were performed using the same general procedure describedfor [^(99m)Tc]SRV32 described in Example 4.

SPECT-CT imaging experiment of compound [¹¹¹In]SRV27 (FIG. 20)illustrated clear PSMA-dependent binding in PSMA+ PC3 PIP xenograftswithin 1 h post injection. The high values noted in kidney are partiallydue to high expression of PSMA within proximal renal tubules (Silver etal., Clin Cancer Res, vol. 3, pp. 81-85, 197; Slusher et al., J CompNeurol, vol. 315, pp. 271-229, 1992). Rapid clearance from the kidneyswas observed while the activity retained in PSMA+tumor even after fourdays post injection.

SPECT-CT imaging experiment of compound [¹¹¹In]SRV100 (FIG. 21)demonstrated similar clear PSMA-dependent binding in PSMA+ PC3 PIPxenografts within 2 h post injection. The high values noted in kidneyare partially due to high expression of PSMA within proximal renaltubules (Silver et al., Clin Cancer Res, vol. 3, pp. 81-85, 197; Slusheret al., J Comp Neurol, vol. 315, pp. 271-229, 1992). Rapid clearancefrom the kidneys was observed while the activity retained in PSMA+tumoreven after four days post injection. The longer tumor activity retentionfor [¹¹¹In]SRV27 and [¹¹¹In]SRV100 might be useful in Y-90/Lu-177 basedradiotherapeutic applications.

FIG. 22 demonstrates clear tumor uptake for [¹¹¹In]SRV73 at 7 h postinjection. This is significant since after attaching a bulky fluorescentdye, rhodamine, the compound retains its PSMA binding activity. This isan example of dual modality application for this class of compounds.

Example 7 SRV134

2-{3-[1-Carboxy-5-(7-{5-carboxy-5-[3-phenyl-2-(3-phenyl-2-{2-[2-(2-tritylsulfanyl-acetylamino)-acetylamino]-acetylamino}-propionylamino)-propionylamino]-pentylcarbamoyl}-heptanoylamino)-pentyl]-ureido}-pentanedioicacid (SRVI34). SRVI34 was prepared according to the scheme below.Lys(Boc)-Wang resin (100 mg. 0.43 mM) was allowed to swell with CH₂Cl₂(3 mL) followed by DMF (3 mL). A solution of 20% piperidine in DMF (3×3mL) was added to the resin that was then shaken gently on a mechanicalshaker for 30 min at ambient temperature. The resin was washed with DMF(3×3 mL) and CH₂Cl₂ (3×3 mL). Formation of free amine was assessed bythe Kaiser test. After swelling the resin in DMF, a solution ofFmoc-Phe-OH (3 eq), HBTU (3 eq), HOBt (3 eq), and DIPEA (4.0 eq) in DMFwas added and gently shaken for 2 h. The resin was then washed with DMF(3×3 mL) and CH₂Cl₂ (3×3 mL). The coupling efficiency was assessed bythe Kaiser Test. That aforementioned sequence was repeated for four morecoupling steps with Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Gly-OH and S-tritylmercaptoacetic acid. Finally the product was cleaved from the resinusing TFA:CH₂Cl₂ (1.1) and concentrated under vacuum to produce the freeamine (SRV132). The concentrated product was purified by using a C₁₈SepPak Vac 2 g column. The product was eluted with a solution 70/30water/acetonitrile (0.1% TFA in each). ESIMS: [M+1]⁺. Lyophilized SRVI32(10 mg, 12 μmol in 2 mL DMF) was added to the urea (compound 1 describedin Example 5) (20 mg, 21.4 μmol in 1 mL DMF) followed by TEA (214 μmol,30 μL) and then stirred at 25° C. for 16 h. The residue was washed 2×5mL CH₂Cl₂ to remove impurities. The colorless solid residue thusobtained was purified by a C₁₈ SepPak Vac 2 g column using an eluent of70/30 water/acetonitrile (0.1% TFA in each). The product was furtherpurified using preparative RP-HPLC by Method 1, retention time 17 min.Yield: ˜ 30%. ESMS m/Z: 1328 [M+H]⁺, ¹H NMR (D₂O/CD₃CN (1:1) δ: 7.98 (m,5H), 7.90-7.76 (m, 18H), 7.66 (m, 2H), 5.11 (m, IH), 4.82-4.72 (m, 3H),4.28 (m, 2H), 4.16 (m, 2H), 3.68 (m, 5H), 3.49-3.32 (m, 2H), 3.00 (m,2H), 2.69 (m, 4H), 2.64-1.74 (m, 26H).

Radiolabeling with Tc-99m: Radiolabeling was performed following aliterature procedure (Wang et al., Nature Protocols, vol. 1, pp.1477-1480, 2006). Briefly, 1 mg (75.3 μmol) of compound SRVI34 wasdissolved in 1 ml of 0.5 M ammonium acetate buffer at pH 8. Disodiumtartarate dihydrate was dissolved in the labelling buffer of 0.5 Mammonium acetate (pH 8) to a concentration of 50 mg/ml. Ascorbicacid-HCl solution was prepared by dissolving ascorbic acid in 10 mM HClto a concentration of 1.0 mg/ml. A solution of SRVI34 (80 μl) wascombined to a solution of 45 μl 0.25 M ammonium acetate, 15 μl tartaratebuffer, followed by 5 μl of the freshly prepared 4 mg/ml SnCl₂. 2H₂Osolution in the ascorbate-HCl solution. The final pH will be about8-8.5. After vortexing, was added 20 mCi of ^(99m)Tc-pertechnetate in200 μl saline and was heated the solution at 90-100° C. for 20 min.Reaction mixture was cooled, diluted 850 μl of water and purified byHPLC using a Phenomenex C₁₈ Luna 10×250 mm² column on a Waters 600EDelta LC system with a Waters 486 tunable absorbance UV/Vis detector,both controlled by Empower software. HPLC solvent system, flow rate=4ml/min, a gradient, 0-5 min, 80/20 water/acetonitrile (0.1% TFA in eachsolvent), 5-25 min 40/60 water/acetonitrile (0.1% TFA in each solvent)and 25-35 min 80/20 (0.1% TFA in each solvent) was used. Tworadiolabeled products were found, called as [^(99m)Tc]SRV134A (5.52 mCi)(retention time 17.5 min) and [^(99m)Tc]SRVI34B (6 mCi) (retention time18.9 min). SRVI34A and SRVI32B are diastereomers, syn and anti-isomerswith respect to the Tc=O group. Each product was neutralized with 50 μlof 1 M sodium bicarbonate and evaporated to dryness under vacuum. Theobtained solid residues was dissolved in 200 μl saline and used forimaging and biodistribution studies.

Ex Vivo Biodistribution

PSMA+ PC-3 PIP and PSMA− PC-3 flu xenograft-bearing SCID mice wereinjected via the tail vein with 30 μCi [^(99m)Tc]SRVI34B. Four mice weresacrificed by cervical dislocation at 30, 60, 120, and 300 min p.i. Theheart, lungs, liver, stomach, pancreas, spleen, fat, kidney, muscle,small and large intestines, urinary bladder, and PC-3 PIP and flu tumorswere quickly removed. A 0.1 mL sample of blood was also collected. Eachorgan was weighed, and the tissue radioactivity was measured with anautomated gamma counter (1282 Compugamma CS, Pharmacia/LKB Nuclear,Inc., Gaithersburg, Md.). The % ID/g was calculated by comparison withsamples of a standard dilution of the initial dose. All measurementswere corrected for decay.

Compound [^(99m)Tc]SRVI34B was assessed for its pharmacokinetics ex vivoin severe-combined immunodeficient (SCID) mice bearing both PSMA+PC3-PIP and PSMA−PC3− flu xenografts (Chang et al., Cancer Res, vol. 59,pp. 3192-3198, 1999). Table 3 shows the percent injected dose per gram(% ID/g) of radiotracer in selected organs for [^(99m)Tc]SRVI34B.

TABLE 3 Biodistribution data for [9^(y)9^(y)m^(m)πTc]SRVI34B (n = 4) 30min 60 min 120 min 300 min Blood 1.13 ± 1.06 0.69 ± 0.08 0.27 ± 0.090.23 ± 0.00 heart 1.11 ± 0.06 0.70 ± 0.16 0.61 ± 0.07 0.46 ± 0.05 lung4.08 ± 0.31 4.84 ± 1.26 4.02 ± 0.73 2.79 ± 0.74 liver 1.55 ± 0.23 0.92 ±0.37  0.50 ± 0.085 0.24 ± 0.09 stomach 0.79 ± 0.23 0.77 ± 0.17 0.54 ±12   0.27 ± 0.08 pancreas 1.72 ± 0.74 1.42 ± 0.45 1.02 ± 0.29 0.94 ±0.46 spleen 56.44 ± 16.49 64.24 ± 13.29 58.27 ± 18.26 24.49 ± 3.63  fat2.18037 ± 2.13 ± 0.58 1.82 ± 0.37 0.99 ± 0.03 0.50 kidney 62.45 ± 1.63 96.38 ± 22.74 104.84 ± 19.03  116.14 ± 2.71  muscle 1.20 ± 0.12 0.74 ±0.04 1.29 ± 1.31 0.45 ± 0.31 small 1.03 ± 0.40 1.43 ± 0.66 0.79 ± 0.330.23 ± 0.12 intestine large 0.61 ± 0.03 0.63 ± 0.38 0.35 ± 0.12 1.30 ±0.08 intestine bladder 1.28 ± 0.25 2.07 ± 0.96 0.87 ± 0.33 0.51 ± 0.00PC-3 PIP 6.11 ± 0.94 7.99 ± 2.26 6.96 ± 1.13 4.81 ± 0.66 PC-3 flu 0.98 ±0.38 0.76 ± 0.51 0.50 ± 0.28 0.22 ± 0.11 PIP:flu 6.28 10.56 14.05 22.18

Small Animal SPECT-CT Imaging

Imaging experiments for [^(99m)Tc] SRVI34A and [^(99m)Tc]SRVI34B weredone following the same procedures as was done for [^(99m)Tc]SRV32(Example 4).

FIGS. 23, 24, 25, and 26 demonstrate the high target selectivity of[^(99m)Tc]SRV134B by delineating the PSMA+tumors. The compound[^(99m)Tc]SRVI34B exhibited high uptake in PSMA+tumor and no uptake inPSMA− tumor. The tumor uptake remains high 4.88% ID/g even at 5 hr postinject (p.i.). However this compound showed very high kidney uptake 116%ID/g even at 5 hr p.i. In addition this compound showed high spleenuptake 24.5% ID/g at 5 hr p.i.

REFERENCES

All publications, patent applications, patents, and other referencesmentioned in the specification are indicative of the level of thoseskilled in the art to which the presently disclosed subject matterpertains. All publications, patent applications, patents, and otherreferences are herein incorporated by reference to the same extent as ifeach individual publication, patent application, patent, and otherreference was specifically and individually indicated to be incorporatedby reference. It will be understood that, although a number of patentapplications, patents, and other references are referred to herein, suchreference does not constitute an admission that any of these documentsforms part of the common general knowledge in the art. In case of aconflict between the specification and any of the incorporatedreferences, the specification (including any amendments thereof, whichmay be based on an incorporated reference), shall control. Standardart-accepted meanings of terms are used herein unless indicatedotherwise. Standard abbreviations for various terms are used herein.

-   Chen Y, Pullambhatla M, Banerjee S, Byun Y, Stathis M, Rojas C,    Slusher BS, Mease RC, Pomper MG. Synthesis and biological evaluation    of low molecular weight fluorescent imaging agents for the    prostate-specific membrane antigen. Bioconjug Chem. 23: 2377-85    (2012);-   Maresca K P, Hillier S M, Femia F J, Keith D, Barone C, Joyal J L,    Zimmerman C N, Kozikowski A P, Barrett J A, Eckelman W C, Babic J W.    A Series of Halogenated Heterodimeric Inhibitors of Prostate    Specific Membrane Antigen (PSMA) as Radiolabeled Probes for    Targeting Prostate Cancer J. Med. Chem. 52: 347-357 (2009);-   Chen Y, Dhara S, Banerjee S, Byun Y, Pullambhatla M, Mease RC,    Pomper MG. A low molecular weight PSMA-based fluorescent imaging    agent for cancer. Biochem. Biophys Res. Commun. 390: 624-629 (2009);-   Pomper, Martin G.; Mease, Ronnie C.; Ray, Sangeeta; Chen, Ying    Psma-targeting compounds and uses thereof;-   International PCT patent application publication no.    WO2010/108125A2, for PSMA-TARGETING COMPOUNDS AND USES THEREOF, to    Pomper et al., published Sep. 23, 2010.-   Rowe, S P, Gorin M S, Hammers H J, Javadi M S, Hawasli H, Szabo Z,    Cho S Y, Pomper M G, Allaf M E. Imaging of metastatic clear cell    renal cell carcinoma with PSMA-targeted ¹⁸F-DCFPyL PET/CT. Ann.    Nucl. Med. 29(10) 877-882 2015.

Although the foregoing subject matter has been described in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be understood by those skilled in the art thatcertain changes and modifications can be practiced within the scope ofthe appended claims.

That which is claimed:
 1. A compound having the structure:

wherein: Z is tetrazole or CO₂Q; each Q is independently selected fromhydrogen or a protecting group; a is 1, 2, 3, or 4; R is eachindependently H or C₁-C₄ alkyl; Ch is a metal chelating moietyoptionally including a chelated metal, wherein Ch optionally includesany additional atoms or linkers necessary to attach the metal chelatingmoiety to the rest of the compound; W is —NRC(O)—, —NRC(O)NR—,NRC(S)NR—, —NRC(O)O—, —OC(O)NR—, —OC(O)—, —C(O)NR—, or —C(O)O—; Y is—C(O)—, —NRC(O)—, —NRC(S)—, —OC(O); V is —C(O)—, —NRC(O)—, —NRC(S)—, or—OC(O)—; m is 1, 2, 3, 4, 5, or 6; n is 1, 2, 3, 4, 5 or 6; p is 0, 1,2, or 3, and when p is 2 or 3, each R¹ may be the same or different; R¹is H, C₁-C₆ alkyl, C₂-C₁₂ aryl, or C₄-C₁₆ alkylaryl; R² and R³ areindependently H, CO₂H, or CO₂R⁴, wherein R⁴ is a C₁-C₆ alkyl, C₂-C₁₂aryl, or C₄-C₁₆ alkylaryl, wherein when one of R² and R³ is CO₂H orCO₂R⁴, the other is H, and when p is 0, one of R² and R³ is CO₂R⁴, andthe other is H.
 2. The compound of claim 1, wherein the compound has thestructure:


3. The compound of claim 1, wherein: Z is CO₂Q; each Q is hydrogen; R isH; a is 4; m is 6; n is 3; p is 2; R¹ is C₂-C₁₂ aryl, wherein the arylmay be substituted or unsubstituted and R¹ may be the same or different;R² is CO₂H; R³ is H; W is —NRC(O)—, wherein R is H; V is —C(O)—; and Chincludes any additional atoms or linkers necessary to attach the metalchelating moiety to the rest of the compound.
 4. The compound of claim3, wherein R¹ is phenyl or a substituted phenyl.
 5. The compound ofclaim 4, wherein R¹ is a phenyl substituted at 1, 2, 3, or 4 positionswith a substituent group selected from the group consisting of halogen,cyano, hydroxyl, nitro, azido, amino, alkanoyl, carboxamido, alkyl,alkenyl, alkynyl, alkoxy, aryloxy, alkylthio, alkylsulfinyl,alkylsulfonyl, aminoalkyl, carbocyclic aryl, arylalkyl, arylalkoxy, anda saturated, unsaturated, or aromatic heterocyclic group, may be furthersubstituted.
 6. The compound of claim 5, wherein R¹ is a phenylsubstituted with a halogen and a hydroxyl.
 7. The compound of claim 1,wherein the additional atoms or linkers necessary to attach the metalchelating moiety to the rest of the compound comprises an alkyl, aryl,combination of alkyl and aryl, or alkyl and aryl groups havingheteroatoms.
 8. The compound of claim 7, wherein the additional atoms orlinkers necessary to attach the metal chelating moiety to the rest ofthe compound comprises an alkyl, wherein the alkyl may be substituted orunsubstituted.
 9. The compound of claim 1, wherein Ch comprises astructure selected from the group consisting of:


10. The compound of claim 1, wherein Ch1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).
 11. Thecompound of claim 1, wherein the compound is selected from the groupconsisting of:


12. The compound of claim 1, wherein Ch includes a chelated metal andthe chelated metal comprises a radioactive isotope.
 13. The compound ofclaim 12, wherein the radioactive isotope is Tc-99m, In-111, Ga-67,Ga-68, Y-86, Y-90, Lu-177, Re-186, Re-188, Cu-64, Cu-67, Co-55, Co-57,Sc-47, Ac-225, Bi-213, Bi-212, Pb-212, Sm-153, Ho-166, or Dy-166. 14.The compound of claim 13, wherein the radioactive isotope is Ga-68 orLu-177.
 15. A method for imaging one or more prostate-specific membraneantigen (PSMA) tumors, or cells the method comprising contacting the oneor more tumors, or cells, with an effective amount of a compound ofclaim 1, and pharmaceutically acceptable salts thereof, and making animage.
 16. The method of claim 15, wherein the imaging comprisespositron emission tomography (PET).
 17. The method of claim 15, whereinthe one or more PSMA-expressing tumors or cells is selected from thegroup consisting of a prostate tumor or cell, a metastasized prostatetumor or cell, a lung tumor or cell, a renal tumor or cell, aglioblastoma, a pancreatic tumor or cell, a bladder tumor or cell, asarcoma, a melanoma, a breast tumor or cell, a colon tumor or cell, agerm cell, a pheochromocytoma, an esophageal tumor or cell, a stomachtumor or cell, and combinations thereof.
 18. A method for treating atumor comprising administering a therapeutically effective amount of acompound of claim 1, where the compound includes a therapeuticallyeffective radioisotope.
 19. A kit comprising a compound of claim 1.